CN112143672B - Pseudomonas aurantiaca CM-7 and application thereof - Google Patents
Pseudomonas aurantiaca CM-7 and application thereof Download PDFInfo
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Abstract
The invention discloses pseudomonas aurantiaca and application thereof. The Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 can obviously inhibit the growth of chestnut blight (cryptosporinitica) and/or ink germ (Phytophthora cinnamon) hyphae and has high-efficiency inhibition effect. The culture condition of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 is simple, easy to store, easy for industrial production and has good development and application prospects.
Description
Technical Field
The invention relates to pseudomonas aurantiaca and application thereof in the technical field of biology.
Background
China is a big agricultural country, and the yield and quality of grains, fruits and vegetables are related to the national stability and the social stability. Economic losses of major crops caused by plant diseases all over the world reach billions of dollars, and 70-80% of the diseases are caused by pathogenic fungi, for example, chestnut blight is caused by the pathogenic fungi causing the chestnut blight.
Chestnut blight bacteria (cryptosporidium parasitica) belong to the order Siberidaceae (Diaporthales) of the class of the ascomyceta (Ascomycota) chaetomycete (Sordariomycetes) and can be parasitic to Castanea sativa, Castanea mollissima, Castanea japonica, Castanea henryi and other quercus species. Chinese chestnut nursery stocks and fruiting trees can be infected by chestnut blight germs, and disease spots can quickly surround branches and trunks after the disease is attacked, so that the yield and the quality of chestnut are obviously reduced, and the whole branches or the whole plant withers when the disease is serious. Chestnut blight bacteria become the most serious bacteria harmful to chestnuts. In 1904, chestnut blight was first discovered in new york, usa, and then in the next half century, the whole american chestnut tree was almost completely destroyed by it. In 1913, chestnut blight was found in China for the first time, and then the chestnut blight was found in all parts of China, and the disease is severe in some areas. The ink disease bacterium (Phytophthora cinnamomea), belonging to the phylum of the phylum isochoryta (anomycota) oomycetes (Oomycota) order Peronosporales (Peronosporales) Peronosporaceae (Peronosporaceae), Phytophthora (Phytophthora), produces an infection that causes "root rot" or "blight" in plants. Plant pathogens are one of the most invasive species worldwide and are present in over 70 countries around the world. The dieback of the plant refers to a plant disease caused by the entry of the lethal Phytophthora cinnamomi (Phytophthora cinnamomi) into the plant body.
Currently, the main method for improving the resistance to chestnut blight and/or ink disease is by traditional breeding, including breeding of disease resistant varieties by crossing American chestnut or European chestnut with resistant Chinese chestnut and Japanese chestnut. In addition, the main control methods for chestnut blight bacteria and/or black fungus bacteria include agricultural control, biological control, chemical control and comprehensive control. Weak virulence bacterial strains containing dsRNA are found in Italy and France, and the harm brought by chestnut blight bacteria can be effectively controlled; part of biological preparations can also play a role in inhibiting; in addition, the carbendazim, the bactericide and other chemical medicines can also play a certain effect.
Pseudomonas (Pseudomonas) is an aerobic group of gram-negative bacteria that have an extremely rich metabolic diversity that also enables them to survive in a very wide niche. Since the middle of the last century, certain species of pseudomonas have begun to be used to inhibit the growth or colonization of crop pathogens. Such applications are commonly referred to as biological control. The biocontrol properties of strains of Pseudomonas fluorescens and Pseudomonas proteins (such as CHAO or Pf-5) are currently the best studied as agents for biocontrol applications and research. Pseudomonas aurantiaca (Pseudomonas aurantiaca) was classified as Pseudomonas chlororaphis, and di-2, 4-diacetylfluoroglobuline produced by Pseudomonas aurantiaca is an antibiotic substance inhibiting gram-positive bacteria. The pseudomonas contains microbial resources which can create great value, has rich biodiversity and is one of the microbial species which produce most bioactive substances. However, the research on the biocontrol activity and biocontrol preparation of pseudomonas citricola is less at present, CN201010247536.5 discloses a pseudomonas citricola strain for promoting plant growth and a culture method thereof, in particular discloses that the pseudomonas citricola strain can promote plant growth, and further discloses application of the pseudomonas citricola strain in preventing and controlling diseases of rape, rice, wheat, tomatoes and corns. CN201110299609.X discloses a preparation method of a plant rhizosphere growth-promoting bacterium Pseudomonas aurantiaca microbial inoculum, and the Pseudomonas aurantiaca microbial inoculum prepared by the method is used for preparing a microbial fertilizer, so that the plant morbidity can be reduced, and the crop growth can be promoted; through an in vitro leaf experiment, the pseudomonas aurantiaca living microbial fertilizer can obviously inhibit plant pathogenic bacteria, enhance the plant resistance to the plant pathogenic bacteria and play a role in preventing and treating the plant pathogenic bacteria. CN201210452050.4 discloses a orange pseudounicellular microbial pesticide and a preparation method thereof, which takes diatomite as a carrier, cultures orange pseudounicellular JD37 bacterial strain to obtain thalli, adds an auxiliary agent of carboxymethyl cellulose solution, and uniformly mixes the carboxymethyl cellulose solution with the processed diatomite to obtain the orange pseudounicellular microbial pesticide, and the biological pesticide can enhance the defense capability of plants to pathogenic fungi and reduce the morbidity of the orange pseudounicellular microbial pesticide.
In conclusion, no report related to the inhibition of chestnut diseases by using an active substance generated by pseudomonas aurantiaca exists so far, and particularly, no report related to the biological control of chestnut blight bacteria and/or ink germs is reported.
Disclosure of Invention
The invention aims to solve the technical problem of how to prevent and treat chestnut blight (cryptonectria parasitica) and/or black fungus (Phytophthora cinnamon) and chestnut diseases caused by the chestnut blight.
Another technical problem to be solved by the present invention is how to control other common and important plant diseases caused by soil-borne fungi and oomycetes pathogenic bacteria, including: the fungi Botrytis cinerea (gray mold caused by Botrytis cinerea), Fusarium oxysporum (blight caused by Fusarium oxysporum), Verticillium dahliae (Verticillium wilt caused by Verticillium dahliae) and the oomycete Phytophthora infestans (late blight caused by Phytophthora infestans).
In order to solve the technical problems, the invention firstly provides a Pseudomonas aurantiaca strain.
The invention provides a Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7, which is registered with CGMCC No.18903 in the China general microbiological culture Collection center. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 11 months and 5 days.
The invention provides a Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 which can grow rapidly on various culture media, including TSA, KB and PDA, the strain growth speed is rapid, and the formed strain colony is orange yellow. The characteristics of easy culture, rapid growth and the like of the Pseudomonas aurantiaca strain (Pseudomonas chlororaphis subsp. aurantiaca) provided by the invention provide convenient conditions for the biocontrol application of the Pseudomonas aurantiaca strain, and provide sufficient material for developing natural active products of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7.
The chestnut blight bacteria inhibitor provided by the invention has the active ingredients of the metabolite of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 or/and the metabolite of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No. 18903.
In the chestnut blight bacteria inhibitor, the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 comprises living cells and/or active metabolites secreted from the cells.
The chestnut blight bacteria inhibitor can also comprise a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane. In the chestnut blight bacterium inhibitor, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The formulation of the chestnut blight bacterium inhibitor can be various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water dispersible granules.
According to requirements, the chestnut blight bacteria inhibitor can be added with surfactant (such as Tween 20, Tween 80, etc.), binder, stabilizer (such as antioxidant), pH regulator, etc.
The invention also provides a method for preparing the chestnut blight bacteria and/or ink germs inhibitor.
The method for preparing the chestnut blight and/or black lead germ inhibitor comprises the steps of culturing the Pseudomonas citricola (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 in a KB culture medium, and collecting fermentation liquor to obtain the chestnut blight and/or black lead germ inhibitor.
The invention also provides any one of the following applications 1) to 4):
1) the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 comprises application of living cells and/or active metabolites secreted from the extracellular space of the living cells in the inhibition of chestnut blight bacteria and/or ink germs;
2) the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 comprises application of living cells and/or active metabolites secreted from the cells in preparation of chestnut blight bacteria and/or ink germ inhibiting agents;
3) the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 comprises living cells and/or active metabolites secreted from the extracellular space of the living cells, and is applied to the preparation of plant disease inhibitors caused by chestnut blight bacteria and/or ink germs;
4) the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 comprises application of living cells and/or active metabolites secreted from the cells to inhibition of plant diseases caused by chestnut blight bacteria and/or ink germs.
The metabolite of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 can be obtained from the fermentation broth of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No. 18903. The metabolite of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 can be prepared by culturing the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 in a liquid culture medium, and removing the cells of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 in the liquid culture medium (fermentation liquid) to obtain the metabolite of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No. 18903.
In the application, the plant diseases are Chinese chestnut epidemic disease and Chinese chestnut ink disease.
In order to solve the technical problems, the invention also provides a method for culturing the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No. 18903.
The method for culturing the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 provided by the invention comprises the step of culturing the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 in a culture medium for culturing Pseudomonas aurantiaca.
The above method for culturing said Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 may comprise the step of culturing said Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 in a liquid medium; the liquid culture medium can be a liquid fermentation culture medium such as a TSA culture medium, a KB culture medium, a PDA culture medium and the like; the culture temperature can be 26-30 ℃, and specifically can be 28 ℃; the culture time is 1-3 days, specifically 2 days.
Experiments prove that the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 can obviously inhibit the growth of chestnut blight bacteria (cryptosporidium parasitica) and/or ink disease bacteria (Phytophthora cinnamon) hyphae and has high-efficiency inhibition effect. The culture condition of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 is simple, easy to store, easy for industrial production and has good development and application prospects.
The invention has the beneficial technical effects that:
1. the invention discovers a Pseudomonas aurantiaca strain for the first time, and the active substances generated by the Pseudomonas aurantiaca strain can inhibit Chinese chestnut diseases, and particularly can be used for biologically preventing and treating chestnut blight bacteria and/or ink germs.
2. The new functional gene in the genome and the new natural active metabolite thereof have important application value. The orange Pseudomonas strain (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 is successfully screened from the soil of the chestnut tree rhizosphere, and the strain has the highest genome similarity (67.86%) with the orange Pseudomonas strain (Pseudomonas chlororaphis subsp. aurantiaca) through genome similarity comparison analysis, so the orange Pseudomonas strain (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 provided by the invention can be used as a new Pseudomonas strain discovered by research.
3. Rhizosphere bacterial strains capable of inhibiting chestnut blight fungus pathogenic bacteria Crypthecothria parasitica and/or ink blight oomycete pathogenic bacteria Phytophthora cinnam can be screened through the confrontation experiment on the in vitro culture medium. The results of the experiments show that the Pseudomonas aurantiaca strain CM7 has strong growth inhibition effect on two pathogenic bacteria simultaneously.
4. In order to further verify the broad-spectrum antibacterial performance of the pseudomonas citricola strain CM7, the control of soil-borne diseases is taken as the key point, and a variety of common and important soil-borne disease fungi and oomycete pathogenic bacteria are selected for testing through the opposite experiment expansion, and the method comprises the following steps: the fungi Botrytis cinerea (gray mold caused by Botrytis cinerea), Fusarium oxysporum (blight caused by Fusarium oxysporum), Verticillium dahliae (Verticillium wilt caused by Verticillium dahliae), and Oomycetes Phytophthora infestans (late blight caused by Phytophthora infestans). The results show that the obtained P.citricola strain CM7 has a broad-spectrum growth inhibitory effect on the growth of selected pathogenic bacteria.
Deposit description
The strain name is as follows: pseudomonas aurantiaca
Latin name: pseudomonas sp.
The strain number is as follows: CM-7
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 11/2019 and 5/month
Registration number of the preservation center: CGMCC No.18903
Drawings
FIG. 1: the contrast graph of the confrontation experiment of the biocontrol bacteria of the invention and fungal pathogenic bacteria Crypthecortia parasitica and oomycete pathogenic bacteria Phytophthora cinnamon;
FIG. 2: the invention relates to a contrast chart of the confrontation experiment of biocontrol bacteria and fungal pathogens Verticillium dahliae, Fusarium oxysporum, Botrytis cinerea and oomycete pathogen Phytophthora infestans;
FIG. 3 is a comparison of the control Pseudomonas aurantiaca strain (DSM19603) against the biocontrol bacteria of the present invention;
FIG. 4: pseudomonas genome analysis map;
FIG. 5: differential analysis of bacterial communities of different chestnut ages, fig. 5 a: performing differential analysis on the communities sampled in 2016; FIG. 5 b: performing colony difference analysis on samples sampled in 2017; FIG. 5 c: OTU distribution of sampled trees of 2016 and 2017; FIG. 5 d: correlation between the variability of bacterial populations (Bray Curtis) and the age difference of each compartment (SO, RH and EC).
FIG. 6: abundance analysis of chestnut core flora, fig. 6 a: analyzing the abundance of the flora in the core soil; FIG. 6 b: analyzing abundance of the flora of the core rhizosphere; FIG. 6 c: and (4) performing flora abundance analysis on the core EC.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The chestnut blight (cryptosporidium parasitica) and/or ink blight (Phytophthora cinnamon) in the following examples are commercially available.
The media used in the following examples are as follows:
TSA medium: 15g of tryptone, 5.0g of soybean peptone, 5.0g of NaCl, 15g of agar powder, pH 7.0-7.2, distilled water l000mL, and moist heat sterilization at 121 ℃ for 20 min.
PDA culture medium: 200g of potato, 20g of glucose, 20g of agar, pH 7.0-7.2, distilled water l000mL, and moist heat sterilization at 121 ℃ for 20 min.
KB medium: 20.0g peptone, 10.0ml glycerol, 1.5g K2HPO4,1.5g MgSO4.7H2O,15g of agar, pH value of 7.0-7.2, distilled water l000mL, and moist heat sterilization at 121 ℃ for 20 min.
Example 1
Isolation and identification of Pseudomonas citricola (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903
1. Isolation of Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903
Collecting a soil sample of the root of a chestnut tree in a Beijing Huairou chestnut garden, obtaining single bacterial colonies of each strain by adopting a gradient dilution plate method, storing in a test tube, carrying out molecular biological identification, and screening to obtain a Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No. 18903.
2. Identification of Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903
(1) Morphological identification
The Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 has rod-shaped thallus, gram-negative staining, no spore, unipolar flagellum, and motility. The bacterial colony can be formed after being cultured on a KB culture medium for 24 hours, and the bacterial colony can produce an orange pigment, is round, has a convex surface, is smooth and viscous, is easy to pick up and has a neat edge.
(2) Molecular identification
Activating a Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp.aurantiaca) CM-7CGMCC No.18903 by using a KB culture medium, picking strain cells from colonies, culturing the strain cells in the KB culture medium for 24 hours (rotating speed: 220rpm) by shaking, collecting the strain cells in a sterile centrifuge tube, and extracting the genomic DNA of the Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp.aurantiaca) CM-7CGMCC No.18903 by adopting the method of a bacterial DNA extraction kit to serve as a template of PCR reaction. 16S rDNA universal primer is used as an amplification primer, wherein the ratio of a forward primer 63F: 5'-CAGGCCTAACACATGCAAGTC-3' and reverse primer 1389R 5'-ACGGGCGGTGTGTACAAG-3'.
The 50 uL PCR reaction system comprises 1 uL template DNA, forward primer P12 uL, reverse primer P62 uL, 2 xTaq PCR MasterMix 25 uL, ddH2O 20μL。
The PCR reaction conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min, 35 cyclic amplifications, extension at 72 ℃ for 10min and preservation at 4 ℃.
Agarose gel electrophoresis is carried out on a PCR amplification product of a 16S rDNA sequence of a Pseudomonas citricola (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 strain to obtain a DNA fragment of about 1470bp, and after sequencing, on-line comparison is carried out on the DNA fragment and a known related sequence in a database by utilizing Geneius 8.1.9 software.
Genomic DNA of Pseudomonas aurantiaca strain CM-7CGMCC No.18903 was subjected to genome re-sequencing simultaneously. The assembly of the whole genome sequence and online comparative analysis on the genome of other known pseudomonas strains were performed by using SPAdes v.3.9.0, and the phylogenetic TREE (fig. 4) was constructed using the bioinformatics tool ModelFinder comprising IQ-TREE, annotated by Python ETE3 library and displayed generated. The genomic similarity of the P.citricola (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 and the P.citricola (Pseudomonas chlororaphis subsp. aurantiaca) strain is the highest (67.86%), while the genomic sequence of the strains CM6, 8-9 has the genomic similarities of 69.32%, 68.72% and 69.26% respectively with the reference P.citricola strain. Therefore, the Pseudomonas strain provided by the invention is identified as a Pseudomonas citricola strain (Pseudomonas chlororaphis subsp. aurantiaca), and the result simultaneously proves that the Pseudomonas citricola strain (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 provided by the invention can be used as a new Pseudomonas strain discovered by research. The new functional gene in the genome and the new natural active metabolite thereof have important application value. Furthermore, the strain (Pseudomonas chlororaphis subsp. aurantiaca DSM19603) is the only strain of this subspecies of DSMZ that can be found, and the genome information is also referred to the genome of this strain.
Experiment one
Effect of the filtrate of Pseudomonas citricola (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 on the inhibition of chestnut blight (Crypthecothria parasitica) and/or ink blight (Phytophtora cinnaon)
The experimental method comprises the following steps: a Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) CM-7CGMCC No.18903 (hereinafter referred to as CM-7 strain) was inoculated into 10mL of a liquid medium culture medium, and shake-cultured at 28 ℃ and 220rpm for 2 days to obtain a suspension of the CM-7 strain. Then, the CM-7 bacterial solution is centrifuged for 5min at 4000rpm, the supernatant is decanted, and the solution is resuspended in 0.9% sodium chloride solution to elute the medium, which is repeated 2-3 times. Adjusting the concentration of the bacterial liquid to about 109Per ml of cell (OD)600＝1.0)。
The chestnut blight (cryptonectria parasitica) and/or the ink germ (Phytophthora cinnamon) are activated by using a PDA plate, and the bacterial cake of the chestnut blight (cryptonectria parasitica) and/or the ink germ (Phytophthora cinnamon) after the activation is obtained by using a puncher with the diameter of 5 mm. 2ul of CM-7 strain is dropped on the circumference 1CM away from the edge of the PDA culture medium, and the hypha of the pathogenic bacteria cake is placed downwards in the center of the PDA culture medium to observe the growth of the pathogenic bacteria. The tests were all set to at least 3 replicates. And the blank control is a PDA culture medium, when the pathogenic bacterial strain hyphae just cover the whole surface on the control PDA culture medium, the lengths of the pathogenic bacterial hyphae on all the culture medium flat plates are measured, quantitative analysis is carried out, and the bacteriostasis rate is calculated.
Wherein, the bacteriostasis rate is [ (control fungus growth radius-treatment fungus growth radius)/control fungus growth radius ] × 100%.
TABLE 1 results of experiments on the confrontation of Pseudomonas aurantiaca and chestnut blight
TABLE 2 results of experiments on the confronting of P.citrifolia strains with ink germs
The experimental results are as follows:
the results of three confronting experiments of the pseudomonas aurantiaca strain CM7 and chestnut blight bacteria in Table 1 show that the pseudomonas aurantiaca strain has obvious inhibition effect on chestnut blight bacteria, and the inhibition rate is 63.56% at least and 72.15% at most. Therefore, the pseudomonas aurantiaca strain CM7 has obvious antagonism on chestnut blight bacteria and can be used as a biocontrol strain.
Table 2 shows that the Pseudomonas aurantiaca CM7 and the ink germs are subjected to three times of confronting experiments, and the results show that the Pseudomonas aurantiaca has obvious inhibition effect on the ink germs, and the inhibition rate is 78.06% at the lowest and 83.47% at the highest. Therefore, the pseudomonas aurantiaca strain CM7 has obvious antagonism on the alternaria philoxeroides and can be used as a biocontrol strain.
As can be seen from the comparison of the confronting experiments of the Pseudomonas aurantiaca strain CM7 and chestnut blight fungus pathogenic bacteria Crypthecothria parasitica and/or Phytophthora cinmamon in figure 1, the Pseudomonas aurantiaca strain CM7 has strong growth inhibition effect on both pathogenic bacteria, and the inhibition effect is obviously better than that of the control group. In order to further excavate the application range of the obtained strain, the method takes soil-borne disease prevention and control as key points, and various common and important soil-borne disease fungi and oomycete pathogenic bacteria are selected for testing through the spreading of a confrontation experiment, and the method comprises the following steps: the fungi Botrytis cinerea (gray mold caused by Botrytis cinerea), Fusarium oxysporum (blight caused by Fusarium oxysporum), Verticillium dahliae (Verticillium wilt caused by Verticillium dahliae) and oomycete Phytohthora infestans (late blight caused by pathogenic mildew). The results show that the obtained Pseudomonas aurantiaca strain CM7 has a broad-spectrum growth inhibition effect on the growth of the selected pathogenic bacteria, as shown in figure 2, and the inhibition effect is obviously better than that of a control group. The 4 strains of Pseudomonas aurantiaca are closely related to two famous biological control agents with antifungal activity, namely P.protegens CHA0 and P.protegens Pf-5. We have further found that these strains exhibit a broad spectrum of antifungal and antibacterial activity and have found by comparison in genomic analysis that they have in their genome a barnanamide biosynthesis gene cluster BGC and a pyrrolidine nitrile BGC. Barnamide is an alicyclic peptide previously identified in another 2 strains of pseudomonas, purified barnamide is antagonistic to certain fungi, and pyrrolidine nitrile is a secondary metabolite produced by several pseudomonas species with strong antifungal activity.
Experiment two
The experimental method comprises the following steps:
this experiment was conducted mainly to compare the control experiment of the P.citricola biocontrol bacteria of the present invention with a control P.citricola (Pseudomonas chlororaphis subsp. aurantiaca DSM19603), and the P.citricola strains CM-6 to 9 selected for testing and a reference strain were inoculated into 10mL of a liquid medium and cultured with shaking at 28 ℃ and 220rpm for 2 days to obtain a suspension of the strains. Then, the bacterial liquid is centrifuged for 5min at 4000rpm, the supernatant is decanted, and the bacterial cells are resuspended with sterile water. This was repeated twice to elute the culture solution. The bacterial suspension was resuspended in sterile water, and the OD600 absorbance was measured to identify the cell concentration of the suspension, which was adjusted to an OD600 of 1.0 (approximately 109 cells/ml of suspension). Activating chestnut blight bacteria and ink blight bacteria by using a PDA (personal digital assistant) flat plate, and obtaining activated chestnut blight bacteria and ink blight bacteria cakes by using a puncher with the diameter of 5 mm. 2ul of reference strains and bacterial liquid of CM-6 to 9 strains are taken and evenly inoculated on the circumference which is 1CM away from the edge of 1/5PDA culture medium, and pathogenic bacteria cake hypha is downwards placed in the center of 1/5PDA culture medium. The culture was incubated at 28 ℃ and the growth of the pathogens was observed. The tests were all set to at least 3 replicates. And the blank control is 1/5PDA culture medium, when the hyphae of the pathogenic strain just cover the whole surface on the 1/5PDA culture medium of the control, the distance from the edge of the hyphae of the pathogenic bacteria to the edge of the bacterial clone on the flat plate of all the culture mediums is measured, the quantitative analysis is carried out, and the difference of the growth inhibition strength of the test strain and the reference strain on the pathogenic bacteria is calculated and compared. Of these, six replicates of the reference strain and three replicates of the test strain.
The experimental results are as follows:
table 3 shows that the orange pseudomonas strain CM6-CM9 and the control orange pseudomonas strain DSM19603 have obvious inhibition effect on chestnut blight bacteria and black ink germs, and the orange pseudomonas strain CM7 has obvious antagonism on the chestnut blight bacteria and the black ink germs and can be used as a biocontrol strain.
As can be seen from the comparison of FIG. 3 between the Pseudomonas aurantiaca CM7 of the present invention and chestnut blight fungus pathogenic bacteria Crypthecortia parasitica and/or Phytophthora cinnaon, the Pseudomonas aurantiaca CM7 has strong growth inhibition effect on both pathogenic bacteria, and the inhibition effect is significantly better than that of the control group.
TABLE 3 results of experiments on the confrontation of Pseudomonas aurantiaca strains of the invention with control strains
Experiment three
Characterization and identification of chestnut tree core microbiota
The experimental method comprises the following steps: in a chestnut garden which is originally built in the Ming dynasty in Beijing in China, chestnut tree soil which is different from decades to hundreds of years is taken as an experimental material, surface soil is removed from a place which is 20-30 cm deep around each tree, and a sample is taken under the edge of a crown. Soil was collected at similar locations and depths, but in places without chestnut roots. Sample collection was repeated 4 times. And (4) carrying out statistical analysis on diversity and abundance distribution in the microbial communities.
Referring to fig. 5, a 2016, one young tree (about 10 years old) and 3 old trees (372, 440, and 620) were sampled. In B2017, 3 young trees (8, 10 and 20 years) and 6 old trees (372, 440, 505, 580, 620 and 830 years) were sampled. OTU distribution was performed on sampled trees in C2016 and 2017. The relative sequence abundance of the phyla of bacteria associated with Soil (SO), Rhizosphere (RH) and Endogenous Compartment (EC) is plotted. The relationship between the variability of the bacterial flora (Bray Curtis) and the age difference of the compartments (SO, RH and EC) was studied. Each point represents the Bray-Curtis dissimilarity (X-axis, Δ years) of the two samples (Y-axis). Regression lines were added by examining the linear relationship between age differences and the associated Bray-Curtis distances. The black cross-hairs represent the average of the brede-cortis in terms of specific age differences.
See fig. 6, core flora of chestnut tree soil, rhizosphere and EC collected in 2016 are plotted in bar graphs (a-c). The first 10 highly abundant OTUs were screened from each fraction to form the core flora. Yellow: the core soil bacteria microbiota contains 20 OTUs; green: the core rhizosphere bacterial microbiota contains 19 OTUs. Blue color: the core EC flora comprises 22 OTUs; bar heights represent the average of the relative abundance of each OTU. Error bars represent the standard deviation of replication. We also compared the microbiota of each compartment with the core flora. This was created by selecting the first 10 OTUs (based on relative abundance) of soil, rhizosphere and EC per tree.
The experimental results are as follows:
comparison of the core microbiota of these 3 partitions for 2016 revealed that these microorganisms were very similar in young and old trees (FIGS. 6 a-c). Notably, pseudomonas (OTU1) was extremely abundant in the rhizosphere of all trees (up to 50%, fig. 6b), while bradyrhizobium (OTU12) was consistently highly abundant in ECs (over 5%, fig. 6 c). For the 2017 trial, the same core microbiome comparison was performed, and the abundant OTUs in soil and root compartment were also very abundant and similar on young and old trees. Pseudomonas OTU1 was again very abundant in the rhizosphere, although the relative abundance was below 2016. The relative abundance of pseudomonas can vary depending on season and year, based on the corn rhizosphere microflora analysis. In this study, the age of these trees was 8 to 800 years or more, indicating that the composition of their core root microbiota is age independent.
Our studies have shown that root microflora of chestnut trees grown in 8-800 years are quite similar. This strongly suggests that castanea mollissima can avoid negative feedback of soil, which contributes to longevity of castanea mollissima. In this study, we found that single pseudomonas OTU was very abundant in chestnut rhizosphere. Such abundant amounts are indicated to be beneficial to their hosts. This OTU has a strong and broad antagonistic activity, one of which can stimulate growth of arabidopsis thaliana (heterologous). Both of these properties contribute to longevity of chestnuts.
Therefore, the screened orange pseudomonas strain beneficial to the growth of the chestnut trees and the core root microbial community formed by the orange pseudomonas strain are floras beneficial to the long-term growth of the chestnut trees. This longevity is genome-dependent, and it appears that tree longevity is positively correlated with an increase in the number of defense-related genes, and plant-soil negative feedback leads to a short lifespan. In addition to defense-related genes, the microbiota of a plant is also important to its growth and health. Through years of research, the microbial community of the ancient chestnut tree in centuries is tested, and the fact that the negative plant-soil feedback is not available is reflected, and the root soil of the ancient chestnut tree contains microorganisms with antagonistic activity on main pathogenic bacteria of chestnut trees, namely the strains screened by the invention.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. Pseudomonas aurantiaca, which is characterized in that: the bacterial strain number of the pseudomonas aurantiaca is CM-7, and the registration number of the pseudomonas aurantiaca in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 18903.
2. Use of Pseudomonas aurantiaca according to claim 1 for the production of biocontrol agents inhibiting Castanea Castaneae (Crypthecothria parasitica), Alternaria auriculata (Phytophthora cinparamon), Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Verticillium dahlia (Verticillium dahlia) and Phytophthora infestans (Phytophthora infestans).
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