CN116925961A - Pantoea agglomerans PxG45 with synergistic Bt protein insecticidal activity and antibacterial effect and application thereof - Google Patents
Pantoea agglomerans PxG45 with synergistic Bt protein insecticidal activity and antibacterial effect and application thereof Download PDFInfo
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- CN116925961A CN116925961A CN202310668438.6A CN202310668438A CN116925961A CN 116925961 A CN116925961 A CN 116925961A CN 202310668438 A CN202310668438 A CN 202310668438A CN 116925961 A CN116925961 A CN 116925961A
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses pantoea agglomerans PxG45 with synergistic Bt protein insecticidal activity and antibacterial effect and application thereof. The PxG strain is plutella xylostella intestinal symbiotic bacteria, has the effects of enhancing insecticidal activity of Bt proteins and inhibiting plant pathogenic fungi such as colletotrichum glomeratum, and is preserved in the microorganism strain preservation center of Guangdong province in 09 of 2021, and the preservation number is GDMCC No:61623 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong. The PxG strain can be used for preparing Bt protein synergistic pesticide, can be used for preparing preparations for preventing and controlling cruciferous vegetable pests and plant pathogenic fungi diseases, and the like, and has the advantage of environmental protection. The invention not only enriches the biocontrol fungus library, but also is beneficial to the prevention and control of cruciferous vegetable pests and plant pathogenic fungi diseases and the development of green novel pesticides.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms. More particularly relates to pantoea agglomerans PxG45 with synergistic Bt protein insecticidal activity and antibacterial effect and application thereof.
Background
The plutella xylostella (Plutella xylostella) is mainly harmful to cruciferous vegetable crops such as carrots, cabbages and broccoli, is one of the most serious insect pests harmful to cruciferous vegetable crops, and the economic loss caused by the plutella xylostella is up to 50 hundred million dollars each year. Bacillus thuringiensis (Bacillus thuringiensis, bt) is used as a microbial source low-toxicity pesticide, can release protoxin after being dissolved in a midgut alkaline environment in an insect body, is then hydrolyzed and activated by protease to form toxin protein (Bt protein) with insecticidal activity, causes symptoms such as intestinal paralysis, perforation, paralysis of insect bodies and the like, then enters a blood cavity for reproduction, causes septicemia, causes death of insect bodies, and has good control effect on cruciferous vegetable pests. However, researches show that the plutella xylostella has serious drug resistance to all current pesticides including Bt proteins, the control is more and more difficult, and a new preparation capable of effectively controlling the plutella xylostella is needed to be developed.
The insect intestinal canal inhabits a large number of intestinal canal symbiotic bacteria, and has important influence on the growth and development of insects, nutrition absorption and organism immunity. In addition, insect gut symbiotic bacteria have been found to have pathogenic bacteria inhibiting effects. For example, zhang Xiaoyu et al have conducted experiments in which isolated and purified symbiotic bacteria of the intestinal tract of solenopsis invicta had been found to have a significant inhibitory effect on the growth of phytopathogenic fungi by Streptomyces sp.DF-5 (Zhang Xiaoyu et al, 2018). With the continuous and intensive research, more and more insect intestinal bacteria with antifungal activity are isolated and purified, for example, streptomyces darkling (Streptomyces canus) BYB02 isolated from termite intestinal tracts can significantly inhibit the growth of rice blast bacteria and apple rot bacteria (Zhang et al, 2013); burkholderia cepacia (Burkholderia cepacia) BsNLG8 isolated from the intestinal tract of brown planthopper significantly inhibited Pyricularia oryzae (Wang et al, 2022). The insect intestinal symbiotic bacteria are an important source for biological control bacteria by virtue of the advantages of environmental protection, no harm to human and livestock, low toxicity, even no toxicity, difficult resistance generation and the like, and the continuous excavation of new insect intestinal symbiotic bacteria is beneficial to the development of novel biological pesticides.
Disclosure of Invention
The invention aims to provide pantoea agglomerans PxG45 with synergistic Bt protein insecticidal activity and antibacterial effect and application thereof.
The first object of the present invention is to provide a strain of Pantoea agglomerans (Pantoea agglomerans) PxG.
The second purpose of the invention is to provide the application of the strain or the culture solution thereof in improving the insecticidal activity of Bt proteins or preparing Bt protein synergistic pesticides.
The third object of the invention is to provide a Bt protein synergistic pesticide.
A fourth object of the present invention is to provide the use of the strain or a culture solution thereof for controlling cruciferous vegetable pests or for preparing a product for controlling cruciferous vegetable pests.
A fifth object of the present invention is to provide a formulation for controlling cruciferous vegetable pests.
A sixth object of the present invention is to provide the use of said strain or a culture broth thereof for inhibiting plant pathogenic fungi or for the preparation of a product for inhibiting plant pathogenic fungi.
A seventh object of the present invention is to provide a phytopathogenic fungi inhibitor.
An eighth object of the present invention is to provide an application of the strain or a culture solution thereof in controlling plant fungal diseases or in preparing a product for controlling plant fungal diseases.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a pantoea agglomerans (Pantoea agglomerans) PxG strain which is separated from the midgut of plutella xylostella and is an intestinal symbiotic bacterium of plutella xylostella, and has the effects of improving the insecticidal activity of Bt protein and inhibiting plant pathogenic fungi such as colletotrichum gloriosa. The pantoea agglomerans (Pantoea agglomerans) PxG strain of the invention is deposited in the microorganism strain collection of Guangdong province at 09 of 2021, and the deposit number is GDMCC No:61623 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong.
The invention claims the application of PxG strain or culture solution thereof in improving the insecticidal activity of Bt protein or preparing synergistic insecticide of Bt protein.
The PxG strain or the culture solution thereof also comprises a preparation prepared from the PxG strain 45 or the culture solution thereof, for example, a microbial inoculum prepared from the PxG strain 45 or the culture solution thereof.
Specifically, the PxG strain can be obtained by centrifugally collecting thalli after amplification culture, and the culture solution of the PxG strain is bacterial solution obtained by culture.
The invention also provides a Bt protein synergistic pesticide, which contains Bt protein and the PxG strain or the culture solution thereof.
The invention also claims the application of the PxG45 strain or the culture solution thereof in controlling cruciferous vegetable pests or preparing products for controlling the cruciferous vegetable pests.
The invention also provides a preparation for controlling cruciferous vegetable pests, which contains Bt protein and the PxG45 strain or a culture solution thereof.
Optionally, the cruciferous vegetable pest is one or more of plutella xylostella, prodenia litura, asparagus caterpillar, cabbage caterpillar, phyllotreta striolata and cutworm.
Specifically, the cruciferous vegetable pest is plutella xylostella.
Specifically, the Bt protein is a Cry1Ac protoxin.
The invention also claims the application of the PxG45 strain or the culture solution thereof in inhibiting plant pathogenic fungi or preparing a product for inhibiting plant pathogenic fungi.
The invention also provides a plant pathogenic fungi bacteriostat, which contains the PxG strain or the culture solution thereof.
Specifically, the plant pathogenic fungi are one or more of colletotrichum (Colletotrichum higginsianum), colletotrichum gloeosporioides (Colletotrichum camelliae), alternaria solani (Alternaria solani), pseudomonas syringae, waxberry pathogenic variant (Pseudomonas syringae pv.myrica), rice blast bacteria (Magnaporthe oryzae) and fusarium oxysporum (Fusarium oxysporum).
Specifically, the fusarium oxysporum is a fusarium oxysporum copal specialization type 4 physiological Race (Fusarium oxysporum f.sp.cube Race 4).
The invention also claims the application of the PxG45 strain or the culture solution thereof in preventing and controlling plant fungal diseases or in preparing products for preventing and controlling plant fungal diseases.
The invention also provides a preparation for preventing and treating plant fungal diseases, and the bacteriostat contains the PxG strain or a culture solution thereof.
In particular, the plant fungal disease is caused by colletotrichum, colletotrichum gloeosporioides, alternaria solani, pseudomonas syringae, bayberry pathogenicity, pyricularia oryzae and/or Fusarium oxysporum.
Specifically, the fusarium oxysporum is a fusarium oxysporum copal specialized type number 4 physiological race.
The invention also provides a method for controlling cruciferous vegetable pests, which is characterized in that a product prepared from PxG strain or culture solution thereof and Bt protein is applied to foods of the cruciferous vegetable pests to attract the cruciferous vegetable pests to eat.
Specifically, the cruciferous vegetable pest is plutella xylostella.
The invention also provides a method for preventing and controlling plant fungal diseases, which comprises the step of spraying an antibacterial agent prepared from PxG strain or culture solution thereof on a pathogenic bacteria infection part.
In particular, the plant fungal disease is caused by colletotrichum, colletotrichum gloeosporioides, alternaria solani, pseudomonas syringae, bayberry pathogenicity, pyricularia oryzae and/or Fusarium oxysporum.
The invention has the following beneficial effects:
the invention provides a pantoea agglomerans (Pantoea agglomerans) PxG45 strain which is separated from the midgut of plutella xylostella and is a plutella xylostella intestinal symbiotic bacterium, has the functions of enhancing the insecticidal activity of Bt protein and inhibiting plant pathogenic fungi such as colletotrichum glomerdianum and the like, and is preserved in the microorganism strain preservation center of Guangdong province in 09 of 2021, wherein the preservation number is GDMCC No:61623 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong. The PxG strain can be used for preparing Bt protein synergistic pesticide, can be used for preparing preparations for preventing and controlling cruciferous vegetable pests and plant pathogenic fungi diseases, and the like, and has the advantage of environmental protection. The invention not only enriches the biocontrol fungus library, but also is beneficial to the prevention and control of cruciferous vegetable pests and plant pathogenic fungi diseases and the development of green novel pesticides.
Drawings
FIG. 1 is a photograph of a colony of strain PxG45 on a plate.
FIG. 2 shows the result of gram staining of strain PxG 45.
Figure 3 shows morphological features of PxG strain under transmission electron microscopy.
FIG. 4 shows the results of phylogenetic analysis of strain PxG 45; the labels on each branch are: genBank serial number + strain name.
FIG. 5 is a graph showing the effect of strain PxG45 and Cry1Ac protoxin on the survival of Plutella xylostella larvae; wherein Cry1Ac+ddH 2 O represents the simultaneous feeding of Cry1Ac protoxin and ddH 2 O; cry1Ac+ PxG45 represents simultaneous feeding of Cry1Ac protoxin and PxG strain; pxG45+ddH 2 O represents a simultaneous feeding PxG45 isolate and ddH 2 O；ddH 2 O is expressed as ddH added alone 2 O，*P<0.05。
FIG. 6 shows the results of an analysis of survival rate of the strain PxG45 on the treatment of the sterile plutella xylostella with the Cry1Ac protoxin; and (3) injection: sterile plutella xylostella larvae n=60; the experiment was performed in three biological replicates, error bars represent standard deviations; the log-rank (Mantel-Cox) test was used, P <0.05.
FIG. 7 shows the results of the antibacterial ability test of strain PxG 45; wherein A is Alternaria hirsuta, B is Alternaria spinosa, C is Fusarium oxysporum Gouba specialized type No. 4 physiological race, D is Alternaria solani, E is Pyricularia oryzae, and F is Pseudomonas syringae Myrica rubra pathogenic variety.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The PxG45 strain is a pantoea agglomerans (Pantoea agglomerans) PxG strain, is separated from the midgut of plutella xylostella and is an intestinal symbiotic bacterium of plutella xylostella; the PxG strain is deposited in the microorganism strain collection of Guangdong province at 09 of 2021, and the strain deposit number is GDMCC No:61623 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong.
Example 1 isolated culture and identification of strains
The isolation culture and identification process of the PxG strain of the invention is as follows:
1. preparation of Selective separation Medium
LB medium: 10g peptone, 5g yeast extract, 10g NaCl,15g agar, dissolved in 1L sterile ddH 2 In O, regulating the pH to 7.0;
nutrient agar medium NA:3g of beef extract, 10g of peptone, 5g of NaCl,15g of agar, dissolved in 1L of sterile ddH 2 In O, the pH was adjusted to 7.0.
2. Isolation of strains
Dissecting Plutella xylostella 3-instar larva in ultra clean bench under aseptic condition, taking out midgut content, centrifuging, and collecting supernatantAnd precipitating, diluting with 10 times 4 concentration gradients, and dipping and diluting with 10 times -4 The multiplied liquids are respectively coated on LB plates and are placed in a constant temperature incubator at 30 ℃ for culture.
3. Continuous purification culture and photographing
After single bacterial colonies grow out of the culture medium, picking the single bacterial colonies according to the color, size and shape of the bacterial colonies, continuously streaking and purifying each single bacterial colony on an LB plate at least for more than 5 times, photographing streak plates of separated bacterial strains, transferring the streak plates into an LB liquid culture medium, shaking the bacterial strains to an exponential growth phase, storing the bacterial strains in 15% glycerol aqueous solution, and freezing the bacterial strains in a refrigerator at a temperature of minus 80 ℃ for later use.
4. Morphological identification of strains
The morphology of strain PxG45 was identified by streaking individual colonies on a plate, gram staining the PxG strain, and observing the internal structure of PxG by ultra-thin section sample preparation method (Haegeman et al 2009) using a transmission electron microscope. As shown in FIG. 1, the plate photograph of the PxG strain obtained by the separation and purification and plate streaking method shows that the bacterial colony of the PxG strain is yellow, the bacterial colony is flat and round, no bulge is found in the middle part, the wet adhesion degree of the bacterial body is high, and the periphery of the bacterial colony is light Huang Yun. The gram-stained photograph of the PxG strain is shown in FIG. 2, and it is clear from FIG. 2 that the bacteria appeared red after gram staining, indicating that it was a gram-negative bacteria. The morphological characteristics of the PxG strain under transmission electron microscope are shown in FIG. 3, and it is clear from FIG. 3 that the PxG strain 45 is a rod-shaped bacterium having no flagella.
5. Molecular characterization of strains
(1) 16S rDNA identification
Extracting the stored genomic DNA of PxG strain by using a bacterial genomic DNA extraction kit (TIANamp Bacteria DNA Kit, tiangen biological company), carrying out PCR amplification by using the extracted DNA as a template and using a 16S rDNA universal primer 27F (5 '-AGTTTGATCMTGGCTCAG-3' and 1492R (5'-GGTTACCTTGTTACGACTT-3'), wherein the reaction system of the PCR amplification is shown in Table 1, after the reaction system is prepared, slightly mixing, centrifuging briefly, and placing on a PCR instrument, wherein the PCR amplification program comprises the steps of pre-denaturation at 98 ℃ for 30S, denaturation at 98 ℃ for 10S, annealing at 55 ℃ for 30S, extension at 72 ℃ for 20S and 35 cycles, and preservation at 72 ℃ for 5min and 4 ℃.
TABLE 1 bacterial 16S rDNA PCR amplification System
The PCR product was subjected to 1% agarose gel detection and gel cutting, recovery and purification, and then was sent to the Guangzhou department of Prime Biotechnology, inc. for sequencing. The 16S rDNA sequence of the PxG strain obtained by the sequencing is shown as follows (SEQ ID NO. 1):
GGGTGGGGCGCTACCTGCAGTCGGACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGGATCTGCCCGATAGAGGGGGATAACCACTGGAAACGGTGGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTCACTATCGGATGAACCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGACGGGGTTAATAACCCTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTTAAGTCAGATGTGAAATCCCCGG GCTTAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGARATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAKATACCCTGGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTTCCCTTGAGGAGTGGCTTCCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAATGAAGTTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACGGAATTTAGCAGAGATGCCTTAGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCACAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTACCATGATTACC
(2) Phylogenetic analysis of strains
Phylogenetic analysis of the strains was performed according to the sequencing results. The sequence was spliced with Chromas software and then blast aligned to the rRNA/ITS database in NCBI, the closest aligned sequence was accession No. NR 041978.1, the sequence was the 16S rna of Pantoea agglomerans strain DSM 3493, and the 16S rDNA sequence of pxg45 strain was 94% similar thereto. In addition, the sequence of the strain PxG is selected, the ClustalW software is used for carrying out multi-sequence comparison and analysis, and the maximum reduction Method (MP) of MEGA 4.0 software is used for carrying out phylogenetic analysis on the sequence with highest similarity between the determined 16S rDNA sequence of the plutella xylostella larva intestinal symbiotic bacteria and part of GenBank database. As shown in FIG. 4, the phylogenetic analysis result of the PxG strain is that the strain PxG and the strain Pantoea agglomerans strain DSM 3493 are gathered together, the homology is higher, the same strain is presumed, and the PxG strain classification unit belongs to the Bacteria; pseudoodonadota; gammaproteicteria; enterobacteriales; erwinicaceae; pantoea; pantoea agglomerans group. From the above results, it can be seen that the strain PxG45 obtained by the screening of the present invention is pantoea agglomerans (Pantoea agglomerans), namely pantoea agglomerans PxG strain, which was deposited with the microorganism strain collection in Guangdong province at 09 of 2021, and the strain deposit number is GDMCC No:61623, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
6. Physiological and biochemical characterization of strains
The physiological and biochemical detection of the PxG strain is carried out by referring to the handbook of identification of common bacterial systems. The physiological and biochemical identification results of the PxG strain are shown in Table 2, and it is apparent from Table 2 that PxG strain can utilize methyl red, fructose, starch, D-mannose, lactose, L-arabinose, D-mannitol, maltose, D-xylose, cellobiose and glucose, and cannot utilize H 2 S, indole, citrate, lactose, raffinose and oxidase.
Table 2PxG physiological and biochemical identification
Note that: "+" is a positive response and "—is a negative response.
Example 2 biological assay of the Cry1Ac protoxin and the PxG Strain on Plutella xylostella
1. Activation culture of PxG strain and preparation of PxG strain suspension
In an ultra-clean bench, 6 mu L of PxG45 glycerol bacteria are inoculated into 6mL of LB liquid medium, and shaking culture is carried out for 12 hours at 30 ℃ by a 200g shaking table; shaking the bacterial liquid according to a proportion of 1:100 (bacterial liquid: culture medium) is inoculated into fresh LB liquid culture medium in proportion, and 200g of the fresh LB liquid culture medium is put into a shaking table to shake for 6 hours at 30 ℃; centrifuging the activated PxG45 bacterial solution at 10,000g for 2min, collecting bacterial cells, and sterilizing with the same volume of sterile ddH 2 O is washed for 3 times, the washed thalli is collected, ddH with the same volume is added 2 O is prepared into bacterial suspension, 200 mu L of bacterial suspension is detected by using an enzyme-labeled instrument, and the OD600 of the bacterial suspension is regulated to be 1.0.
2. Breeding of plutella xylostella
The breeding conditions of the plutella xylostella (DBM-W) are as follows: temperature: 25.+ -. 1 ℃ photoperiod: 16h:8h (light: dark), relative humidity: 75%. The plutella xylostella larvae are fed with artificial feed, and the formula of the artificial feed is as follows: 40 g of yeast powder, 75 g of wheat germ powder, 2 g of compound vitamin, 2 g of sorbic acid, 2 g of nipagin, 2 g of ascorbic acid, 20 g of sucrose, 6 g of radish seed, 12 g of agar, 2 ml of rapeseed oil, 3-4 drops of linoleic acid and 500 ml of water; the adults are fed with 10% of honey water to supplement nutrition, and eggs are collected by egg cards immersed in the radish juice.
3. Cry1Ac protoxin extraction method
(1) Activating bacillus thuringiensis (Bt) HD73 glycerol bacteria to obtain seed liquid, streaking the seed liquid on an LB solid culture medium plate, after single colony grows out, picking the single colony into an LB liquid culture medium, culturing for 12 hours at 220rpm and 30 ℃, and then culturing for 1:100 (bacterial liquid: culture medium) the bacterial liquid is transferred to 1/2LB culture medium in proportion, 220rpm,30 ℃ for 28.5h;
(2) Centrifuging at 4 ℃ for 10 minutes at 8000g, and collecting precipitate;
(3) 1M NaCl (precooling) 1 time, ddH 2 O (sterilizing and precooling) cleaning for 1 time;
(4) By Buffer A (50mM Na 2 CO 3 ，50mM EDTA-Na 2 3% beta-ME, pH 10.0), adjusting pH to 9.6-10, 100g, incubating on ice for 1h;
(5) Centrifuging at 4deg.C for 20min at 13000g, and collecting supernatant;
(6) The collected supernatant was incubated with 4M NaAc-HAc at a pH adjusted to 4.5,4 ℃overnight;
(7) Centrifugation at 13000g at 4℃for 15min, ddH 2 O was washed 2 times with 50mM Na 2 CO 3 (PH 9.6) dissolution;
(8) ddH is used for Cry1Ac protoxin stock solution 2 Diluting O to 20mol/L;
(9) Every 1g of feed is mixed with 250 mu L of sample liquid, starvation treatment is carried out for 4 hours before the larvae of plutella xylostella 3 years old are fed, and the number of living insects is counted every 12 hours from 24 hours after feeding until 84 hours.
4. Toxicity determination of Cry1Ac protoxin on plutella xylostella
Sterile ddH for Cry1Ac protoxin extracted by the steps 2 O was diluted to 7 different concentrations (5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL, 200. Mu.g/mL) while a set containing only sterile ddH was provided 2 O served as a blank, 4 replicates per treatment, 10 replicates per treatment. 1g of artificial feed was added to each replicate in a sterile petri dish (diameter 9 cm), 250. Mu.L of Cry1Ac protoxin dilutions of different concentrations were added dropwise to the feed per dish, and additionally sterile ddH was added 2 One group of O served as a blank. After stirring evenly, 10 plutella xylostella larvae which are about 4 hours old and are subjected to starvation treatment are respectively picked, and after the picking, the plutella xylostella larvae are transferred into a artificial climatic chamber for breeding, and conditions such as temperature and humidity of the climatic chamber are consistent with those of normal breeding. After 36h, the number of live insects in each dish was counted and the mortality corresponding to each treatment was calculated, and the corrected mortality for each treatment was obtained using the Abbott formula (Abbott, 1987) with reference to the mortality case of the control group, and then the SPSS 22 was used for the Probit analysis and the slope of the virulence regression equation, sub-lethal dose (LC 25) and its 95% confidence interval (Finney, 1971) were calculated.
TABLE 3 determination of sensitivity of Plutella xylostella 3-instar larvae to Cry1Ac protoxins
5. Treatment of plutella xylostella with Cry1Ac protoxin
160 plutella xylostella 3-instar larvae which grow healthily and have consistent instar are randomly selected, and starvation treatment is carried out for 3 hours. Dividing the treated larvae into 4 groups, and dividing each group into 4 subgroups as repeated groups, wherein 10 plutella xylostella 3-instar larvae are arranged in each repeated group; the formulation of the 4 sets of treatments is specifically as follows:
group 1: with 125. Mu.L of an intestinal PxG strain suspension (OD 600 of 1.0) and 125. Mu.L of ddH 2 O and 1g of feed are added to feed 3-instar larvae;
group 2: mu.L of enterobacteria PxG and 45 bacterial suspension (OD 600 is 1.0) is added with 125 mu.L of 20mol/L Cry1Ac protoxin and 1g of feed to feed 3-year larvae;
group 3: 125 mu L of 20mol/L Cry1Ac protoxin plus 125 mu L of ddH 2 O and 1g of feed are added to feed 3-instar larvae;
group 4: with 250. Mu.L ddH 2 O and 1g of feed are added to 3-instar larvae.
The number of surviving plutella xylostella insects was recorded every 12h, until 96h, data processing and chart making were performed using Graphpad 8.0 software, and difference significance analysis was performed using Student's-T test.
The effect of PxG strain and Cry1Ac protoxin on the survival rate of plutella xylostella larvae is shown in fig. 5, and the result shown in fig. 5 shows that after PxG bacterial suspension and Cry1Ac protoxin are mixed and added for 3d, the survival rate of plutella xylostella is 37.0963% and the survival rate after 4d addition is 9.27406%; after 3d of independent feeding of Cry1Ac protoxin, the survival rate of plutella xylostella is 58.2349.1%, after 4d of independent feeding is 51.272%, after 4 days of independent feeding of PxG strain 45, the survival rate of plutella xylostella is 84.4581%, which shows that PxG strain 45 has remarkable synergism on Cry1Ac protoxin, and reaches a remarkable difference level (P < 0.05).
EXAMPLE 3 synergistic Effect of PxG Strain on plutella xylostella Cry1Ac protoxin
1. Preparation of antibiotic solutions
1mg/mL ciprofloxacin, 1mg/mL levofloxacin and 2mg/mL metronidazole were formulated separately.
2. Collecting egg cards and sterilizing
Collecting egg cards produced in the oviposition full period of plutella xylostella, sterilizing in 5 per mill sodium hypochlorite disinfectant for 10 minutes, soaking in clear water twice for 5 minutes each time, sucking the egg cards with water-absorbing paper, placing the egg cards in an insect-raising box, and adding feed for feeding after the insects are hatched for 1-2 days.
3. Antibiotic feeding treatment
Weighing 4 g of plutella xylostella artificial feed in a sterile plastic cup, then adding more than 333 mu L of the prepared ciprofloxacin, levofloxacin and metronidazole antibiotic solution as treatment groups respectively, uniformly stirring, taking a proper amount of the mixture, placing the mixture in an insect raising box, replacing fresh feed once a day, continuing to feed 3-year old larvae from the first hatched larvae, and feeding normal feed 24 hours before treatment; the control group was fed normal feed all the time from the start of hatching.
4. Detection of intestinal bacteria removal effect
The treatment group and the control group were randomized to 10 insects, and the midgut contents were dissected and aseptically ddH extracted 2 O gradient dilution 10 3 ～10 4 100 mu l of coated LB solid medium (without resistance) is taken and placed in a 30 ℃ incubator for cultivation, and after 48 hours, the presence or absence of bacterial growth is detected.
5. Isolated bacterium for restoring and supplementing intestinal tract
Taking out the intestinal tract isolated bacterium glycerol bacteria with the strain identification, removing the repeated strains, and respectively carrying out the steps of 1:1000 proportion is transferred into LB liquid culture medium, seed bacteria are obtained by overnight activating culture, and the seed bacteria are prepared according to the following steps: 100 was transferred to fresh LB medium for about 6h. Centrifuging the bacterial liquid after culturing, enriching bacterial cells, and sterilizing with ddH 2 O was repeatedly washed 3 times to remove the residual medium, and the cells were subjected to ddH 2 O was diluted to OD after resuspension 600 1.0 g of plutella xylostella artificial feed is weighed and placed in a 90mm sterile culture dish, 250 mu L of the diluted bacterial suspension is dripped into the dish, and 10 head three-year-old plutella xylostella sterile larvae are picked into the dish after the mixture is stirred uniformly.
The isolated plutella xylostella enteric symbiotic PxG strain was subjected to anaplerotic supplementation on the intestinal free strain population, and the sensitivity of the intestinal free strain to Cry1Ac protoxin was measured, and the result is shown in FIG. 6. As can be seen from fig. 6, the anaplerosis of the PxG strain can significantly improve the sensitivity of the plutella xylostella larva to the Cry1Ac protoxin (P <0.0001, log-rank (Mantel-Cox) test), and the anaplerosis of the PxG strain reduces half-life cycle of plutella xylostella (Median Survival Time), so that the half-life cycle of plutella xylostella is reduced from 2.5 days to 1.5 days.
EXAMPLE 4 inhibition Activity of the PxG Strain 45 against phytopathogenic fungi
1. Preparation of PxG and 45 bacterial liquid
Glycerol bacteria stored at-80 ℃ were treated with 1:100 is inoculated with fresh liquid LB culture medium, 220rpm, and activated for 12 hours at 37 ℃ to obtain seed bacteria, and then 1:100 is inoculated in fresh LB culture medium, 220rpm and shaking for 4h at 37 ℃ to obtain bacterial liquid with stronger activity, and OD value is measured to prepare 1X 10 7 CFU/mL bacterial liquid.
2. PxG45 bacteriostatic ability detection
The bacteriostatic ability of PxG strain against plant pathogenic fungi was measured by plate counter method (Fernandes et al 2021), activated plant pathogenic fungi cake was inoculated in PDA medium center, bacterial liquid of PxG strain was streaked at a symmetry position 2.5cm from the cake, and the bacterial liquid was inverted cultured with plate inoculated with pathogenic fungi alone as a control at 25±1 ℃ and 75±5% humidity, and colony diameters of the treated group and the control group were measured according to the growth cycle of pathogenic fungi, and the bacteriostatic action of PxG strain was evaluated. The plant pathogenic bacteria to be tested are colletotrichum (c. Higgins ianum), colletotrichum (c. Camelliae), alternaria solani (a. Solani), pseudomonas syringae, bayberry pathogenic variety (p. Syringae pv. Myrica), fusarium oxysporum, copperleaf No. 4 physiological Race (f. Oxysporum f. Sp. Cubic wave Race 4) and rice blast fungus (m. Oryzae).
Inhibition% = [ (pathogen colony diameter in control group-pathogen colony diameter in treatment group)/(pathogen colony diameter in control group) ] ×100%.
3. Experimental results
As shown in FIG. 7, the antibacterial ability test result of the PxG strain is shown in FIG. 7, and the PxG strain can significantly inhibit the growth of colletotrichum (C.higgins ianum), colletotrichum (C.camelliae), alternaria solani (A.solani), pseudomonas syringae, myrica rubra, pyricularia oryzae (M.oryzae) and Fusarium oxysporum Gouba specialization No. 4 physiological Race (F.oxysporum f.sp.cube Race 4).
The diameter of the colletotrichum glomeratum after 8d culture in a culture dish is 48.84 plus or minus 0.73mm, and the diameter of the colletotrichum glomeratum after 8d culture in the culture dish is 24.93 plus or minus 0.33mm with the PxG strain; the diameter of the colletotrichum gloeosporioides after 7d culture in a culture dish is 57.43 plus or minus 0.42mm, and the diameter of the colletotrichum gloeosporioides after 7d culture in the culture dish with PxG strain is 28.71 plus or minus 0.19mm; the diameter of the Fusarium oxysporum Guba specialization No. 4 physiological race after 5d culture in a culture dish is 74.55 +/-0.84 mm, and the diameter of the Fusarium oxysporum Guba specialization No. 4 physiological race after 5d culture in the culture dish and the diameter of the Fusarium Guba specialization strain PxG strain after 5d culture in the culture dish are 34.96 +/-0.39 mm; the diameter of Alternaria solani after 6d culture in the culture dish is 73.45 +/-0.40 mm, and the diameter of Alternaria solani after 6d culture in the culture dish is 33.48+/-0.17 with PxG strain; the diameter of the rice blast bacteria after 7d culture in the culture dish is 47.91 +/-0.61 mm, and the diameter of the rice blast bacteria after 7d culture in the culture dish is 23.44+/-0.74 mm with the PxG strain; the diameter of the pathogenic variant of the pseudomonas syringae waxberry after 6d culture in a culture dish is 83.45 +/-0.22 mm, and the diameter of the pathogenic variant of the pseudomonas syringae waxberry after 6d culture in the culture dish is 83.45 +/-0.22 mm; the results are substituted into a bacteriostasis rate calculation formula to calculate, and the PxG strain shows strong bacteriostasis effect on the 6 pathogenic fungi, and the bacteriostasis rate is more than 49%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A strain of pantoea agglomerans (Pantoea agglomerans) PxG, characterized in that said strain was deposited at the cantonese province microorganism strain collection at 09 of 2021, under the accession number GDMCC No:61623.
2. the use of the strain or the culture solution thereof in improving insecticidal activity of Bt protein or preparing synergistic insecticide of Bt protein.
3. A Bt protein synergistic insecticidal composition, wherein said insecticidal composition comprises Bt protein and the strain of claim 1 or a culture broth thereof.
4. The use according to claim 2 or the pesticide according to claim 3, wherein the Bt protein is a Cry1Ac protoxin.
5. Use of the strain according to claim 1 or a culture solution thereof for controlling cruciferous vegetable pests or for the preparation of a product for controlling cruciferous vegetable pests.
6. A formulation for controlling cruciferous vegetable pests, comprising Bt protein and the strain of claim 1 or a culture broth thereof.
7. The use according to claim 5 or the formulation according to claim 6, wherein the cruciferous vegetable pest is plutella xylostella.
8. The use of the strain or the culture solution thereof according to claim 1 for inhibiting plant pathogenic fungi or for preparing a product for inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are one or more of colletotrichum gloeosporioides, alternaria solani, pseudomonas syringae, myrica grisea, pyricularia oryzae and Fusarium oxysporum.
9. A plant pathogenic fungi bacteriostat, characterized in that the bacteriostat contains the strain or the culture solution thereof according to claim 1; the plant pathogenic fungi are one or more of colletotrichum gloeosporioides, alternaria solani, pseudomonas syringae, myrica rubra, pyricularia oryzae and Fusarium oxysporum.
10. Use of a strain according to claim 1 or a culture solution thereof for controlling plant fungal diseases or for the preparation of a product for controlling plant fungal diseases, characterized in that the plant fungal diseases are caused by colletotrichum gloeosporioides, alternaria solani, pseudomonas syringae, myrica grisea, pyricularia oryzae and/or fusarium oxysporum.
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