CN115927043B - Pseudomonas palehrli Long Nishi strain ZJUCS1001 and application thereof - Google Patents
Pseudomonas palehrli Long Nishi strain ZJUCS1001 and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the field of microorganisms, in particular to pseudomonas strain-pseudomonas paler Long Nishi pseudomonas (Pseudomonas palleroniana) ZJUCS1001 and a prevention and treatment effect thereof on tea tree anthracnose. Pseudomonas palustris (Pseudomonas palleroniana) strain ZJUCS1001 with a preservation number of CGMCC NO:24722. use of pseudomonas paler Long Nishi strain ZJUCS1001, at least any one of the following: inhibit tea tree anthracnose and prevent and treat tea tree anthracnose.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to pseudomonas strain-pseudomonas paler Long Nishi pseudomonas Pseudomonas palleroniana ZJUCS1001 and a prevention and treatment effect thereof on tea tree anthracnose.
Background
Tea tree endophyte (tea plant endophytic bacteria) refers to all bacteria that can colonize the tea tree body at some point in its life without significant damage to the host, and belongs to a prokaryote. The tea tree endophytes can be obtained by separating the roots, stems, leaves, flowers and fruits of various tea trees. Under the action of long-term symbiotic evolution, a good mutual-benefit win-win relationship is established between endophytes and hosts. On the one hand, endophytes obtain nutrients from the plant body which are necessary for life, and on the other hand, endophytes have a beneficial effect on the physiological activity of the host. Endophytes can antagonize plant pathogenic bacteria by competing with the space of the nutrients, producing secondary metabolites against pathogenic bacteria, inducing expression of host defense related genes, and the like, thereby reducing the occurrence rate of plant diseases. Furthermore, endophytes have the advantage of not causing infection or disease in the host, and thus are potential candidate strain sources for biocontrol applications.
The invention of 202111428354.2, namely pseudomonas palustris Long Nishi and application thereof in soil-borne disease control of oomycetes pathogenic bacteria, discloses a separated pseudomonas palustris Long Nishi (Pseudomonas palleroniana), and the preservation number is CCTCCNO: M2020821. The preparation method can be used for preparing medicines for preventing and treating soil-borne diseases of crop oomycetes, wherein pathogenic bacteria are Pythium aphanidermatum (Pythium aphanidermatum), phytophthora nicotianae (Phytophthora nicotianae), phytophthora capsici (Phytophthora capsici), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), rhizoctonia solani (Rhizoctonia solani), colletotrichum gloeosporium (Colletotrichum gloeosporioides) and/or Botrytis cinerea.
Disclosure of Invention
The invention aims to solve the technical problem of providing a novel pseudomonas strain Paylor Long Nishi pseudomonas Pseudomonas palleroniana ZJUCS1001 and application thereof, wherein the strain can prevent and treat anthracnose of tea trees.
In order to solve the technical problems, the invention provides a pseudomonas paler Long Nishi (Pseudomonas palleroniana) strain ZJUCS1001 with a preservation number of CGMCC NO:24722.
the deposited information for strain ZJUCS1001 is as follows: preservation name: pseudomonas palettes Long Nishi Pseudomonas palleroniana, deposit unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4 months, 19 days, deposit number: CGMCC NO:24722.
the invention also provides application of the strain, at least any one of the following: inhibit tea tree anthracnose and prevent and treat tea tree anthracnose.
As an improvement of the use of the invention: inhibiting the growth of hyphae of tea tree anthracnose germ (Colletotrichum camelliae) and reducing the infection rate of tea tree leaf anthracnose.
The strain has the following beneficial effects:
1. the hypha growth inhibition rate of the tea tree anthracnose germ (Colletotrichum camelliae) reaches 71.31 percent.
2. The effect of the pseudomonas paler Long Nishi strain on preventing and controlling anthracnose of tea trees reaches 85.14 percent.
3. Effectively reduces the anthracnose infection rate of tea leaves by 48.08 percent.
It should be emphasized that: the colletotrichum gloeosporioides (Colletotrichum gloeosporioides) is different from the camellia anthracis (Colletotrichum camelliae) which is a tea tree colletotrichum pathogen in the invention: the colletotrichum gloeosporioides (Colletotrichum gloeosporioides) are complex species and contain a large number of anthrax species. The camellia anthracnose (Colletotrichum camelliae) is a dominant pathogenic species of tea tree anthracnose in China, has certain differences with other gum cell anthracnose strain forms and the like, and is mainly expressed in the following aspects: the colony of the camellia anthracnose (Colletotrichum camelliae) is approximately round, the conidium is colorless and smooth, the conidium is mono-spore, oblong or cylindrical, the circle at two ends or one end is slightly thick, the other end is slightly pointed, the size is (13+/-3.5) mu m× (3+/-2) mu m, the attached spore is generated from hypha, the tail end is expanded, the shape is irregular, and the color is deeper. The colletotrichum gloeosporioides (Colletotrichum gloeosporioides) is on the PDA culture medium, and the colony is round; the conidiophore discs are black or dark brown, round or oval, conidiophore cylinders or oblong, sometimes slightly smaller at one end, and the unit cells are colorless, with an oil sphere, the spore size (15.10.+ -. 4.6) × (5.48.+ -. 0.63) μm. The two are different in pathogenicity, and camellia anthracnose (Colletotrichum camelliae) has the strongest pathogenicity on tea leaves. Thus, the corresponding control agents are not identical. That is, a microbial agent having an inhibitory effect on colletotrichum gloeosporioides (Colletotrichum gloeosporioides) may not have an effective effect on Colletotrichum camelliae.
The camellia anthracnose (Colletotrichum camelliae) is a dominant pathogenic species of tea anthracnose in China, and causes great loss to tea production every year. The pseudomonas paler Long Nishi (Pseudomonas palleroniana) separated by the invention has 71.31 percent of inhibition effect on the growth of hyphae of camellia anthracnose (Colletotrichum camelliae), and can effectively prevent the occurrence of tea leaf anthracnose. However, the invention of 202111428354.2, namely pseudomonas palustris Long Nishi and application thereof in soil-borne disease control of pathogenic bacteria of the class oomycetes, only researches that the hypha growth inhibition effect of pseudomonas palustris Long Nishi (Pseudomonas palleroniana) on colletotrichum gloeosporioides (Colletotrichum gloeosporioides) is only 51.41 percent, and does not report the control effect on tea tree anthracnose.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a graph showing the results of a Pseudomonas pauciflora plate inhibition experiment,
compared with a control CK (left), the pseudomonas palustris Pseudomonas palleroniana ZJUCS1001 (right) strain has a certain effect (middle) of inhibiting the growth of the camellia anthracis Colletotrichum camelliae of the tea anthracis on the WA plate on inhibiting the growth of the camellia anthracis Colletotrichum camelliae of the tea anthracis;
FIG. 2 shows an antibacterial and antiviral experiment of Pseudomonas pahler Long Nishi on tea trees, inoculating fungus Colletotrichum camellia of tea tree anthracnose, pseudomonas pahler Pseudomonas palleroniana ZJUCS1001 of Pseudomonas pahler Long Nishi and Pseudomonas sp. Sp strain of the same genus in the invention, inoculating fungus Colletotrichum camellia of tea tree anthracnose on leaves, and simultaneously inoculating Pseudomonas palleroniana ZJUCS1001 strain to reduce the incidence rate of leaf anthracnose of tea tree.
FIG. 3 is a statistical chart of the incidence of the antibacterial and antiviral experiment of Pseudomonas pauciflora on tea trees. Wherein the incidence of anthracnose of tea leaves of the control group is 56.47%, the incidence of Pseudomonas sp. Strain inoculation is 31.59%, and the incidence of Pseudomonas sp. Strain inoculation is Pseudomonas palleroniana ZJUCS 1001.1001 is 8.39%.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
EXAMPLE 1 isolation of Pseudomonas strain Parler Long Nishi Pseudomonas Pseudomonas palleroniana ZJUCS1001 Strain
Nutrient agar medium (NA): the formulation includes peptone 10.0g; 5.0g of beef extract powder; 5.0g of sodium chloride; 12.0g of agar; ph=7.3, distilled water to volume to 1000mL; the preparation method comprises weighing 10.0g peptone, 5.0g beef extract powder, adding 12g agar according to actual experiment requirement, continuously heating, stirring, mixing, adding 5.0g sodium chloride after agar is dissolved, stirring, cooling slightly, supplementing water to 1000mL, regulating pH to 7.3, packaging into test tubes or conical flasks, and plugging and packaging. Sterilizing at 121 deg.c for 20 min at 1.1 atm, cooling and storing.
The strain of the present invention was isolated and cultured in a laboratory under the following conditions.
Pseudomonas paler Long Nishi strain Pseudomonas palleroniana ZJUCS1001 is isolated from tea leaves of Longjing 43 variety of tea plant at the university of Zhejiang in Hangzhou, zhejiang province, tea laboratory base (30.39N, 119.88E). The collected leaf samples were taken back to the laboratory and placed under running water for rinsing to remove surface impurities. Surface disinfection is performed after the rinsing is completed.
After surface sterilization, a proper amount of plant material was taken and placed in a sterile mortar to be ground to a homogenate, then the homogenate was subjected to gradient dilution with sterile water, and the diluted homogenate was spread on NA medium and placed in an incubator to be cultured for 24 hours. Single colony is picked up in a sterile super clean bench, streaked and separated three times to obtain a pure culture.
Endophytic bacterium ZJUCS1001 with growth inhibition effect on camellia anthracis Colletotrichum camelliae is selected as pseudomonas strain paler Long Nishi pseudomonas selected by the invention through a plate counter experiment.
The morphological characteristics of the strain are as follows: circular, yellowish white colonies were presented on NA plates. After incubation of gram-negative bacteria on NA medium for 48 hours at 25℃the bacteria formed translucent Huang Baise round raised colonies with diameters of 2-3mm, with smooth surfaces and clean edges.
Example 2 identification of Pseudomonas paler Long Nishi Pseudomonas palleroniana ZJUCS1001
1. Molecular identification:
1) PCR amplification of bacterial 16s rDNA Gene
PCR amplification was performed using a 50. Mu.L reaction system containing: the upstream and downstream primers were 2. Mu.M each, dNTPs 200. Mu.M, mg 2+ 1.5mM,10 XPCR buffer 5. Mu.L, bacterial liquid 2. Mu.L, taq enzyme 2U, ddH 2 O was made up to 50. Mu.L. Upstream primer 16S sequence5'-AGAGTTTGATCMTGGCTCAG-3' and the downstream primer sequence 5'-TACGGYTACCTTGTTACGACTT-3'. The PCR amplification reaction was performed on a BIORAD S1000 type PCR apparatus.
Reaction conditions: pre-denaturation at 95 ℃ for 5min, followed by 35 cycles, including: denaturation at 95℃for 30sec, annealing at 55℃for 30sec, and extension at 72℃for 1min. Finally, the mixture was extended at 72℃for 7min.
2) And (3) recovering and purifying a PCR product:
after the PCR reaction was completed, the PCR product was detected by 1% agarose gel electrophoresis, and then was performed using a DNA gel purification kit of Axygen Biotechnology, inc., according to the procedures described in the kit.
The method comprises the following steps:
(1) After electrophoresis, the gel containing the target DNA fragment was cut with a blade under an ultraviolet lamp, placed in a 2mL centrifuge tube, and weighed.
(2) 3 volumes of DE-A buffer were added and incubated at 75℃for 10min, during which time shaking was performed several times, until complete thawing was achieved.
(3) Adding 0.5 times of DE-B buffer solution, and mixing uniformly.
(4) The DNA preparation tube was placed in a 2mL centrifuge tube, the mixture was transferred to the DNA preparation tube, centrifuged at 12000rpm for 1min, and the supernatant was discarded.
(5) The DNA preparation tube was placed in a 2mL centrifuge tube, and 500. Mu.L of buffer W1 was added thereto and centrifuged at 12000rpm for 30s.
(6) The DNA preparation tube was placed in a 2mL centrifuge tube, and 700. Mu.L of buffer W2 was added thereto, and the mixture was centrifuged at 12000rpm for 30s.
(7) Repeating the step (6) once.
(8) The DNA preparation tube was placed back into a 2mL centrifuge tube and centrifuged at 12000rpm for 2min. To remove the washing liquid from the dry film.
(9) The DNA preparation tube was placed back into a 2mL centrifuge tube and 50. Mu.L of ddH was added 2 O, centrifugation at 10000rpm for 1min, and storing the eluted DNA at-20deg.C.
3) Sequencing and sequence analysis of genes
And (3) delivering the purified and recovered target DNA fragment to an abiprisa 377 type automatic sequencer for sequencing by a Shanghai worker after electrophoresis detection. After the sequencing result is strictly checked, a 1407bp DNA fragment sequence (SEQ ID NO:1 of the sequence list) is obtained. The nucleotide sequences thus determined were searched and aligned in GenBank using BLAST for homologous or similar nucleotide sequences. The species of the strain under study was judged based on database annotation of homologous sequences, in combination with the morphological structure of the strain.
Through BLAST comparison, the coverage rate of the sequence with the accession number KJ767367.1 is 99%, the similarity is 100%, the coverage rate of the sequence with the accession number JQ770187.1 is 99%, the similarity is 100%, and the coverage rate of the sequence with the two sequences is higher through comparison. The bacterium was thus identified as Pseudomonas paler Long Nishi.
The deposited information for strain ZJUCS1001 is as follows: preservation name: pseudomonas palettes Long Nishi Pseudomonas palleroniana, deposit unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4 months, 19 days, deposit number: CGMCC NO:24722.
strain Pseudomonas palleroniana ZJUCS1001 of the present invention is classified as follows: bacterial kingdom (bacteriosis), proteobacteria phylum (Proteobacteria), gamma-Proteobacteria (Gamma-Proteobacteria), pseudomonas (Pseudomonas) of the family Pseudomonas (Pseudomonas) and Pseudomonas (Pseudomonas).
Example 3: inhibition of tea tree anthrax fungus (Colletotrichum camelliae) hypha growth by pseudomonas palettes Long Nishi Pseudomonas palleroniana ZJUCS1001
Description: in the isolation of the strain described in example 1, a control strain of Pseudomonas sp was also obtained, which has an inhibitory effect on the hyphal growth of tea tree anthrax fungus (Colletotrichum camelliae). The same procedure as in example 2 was used, based on 16S rDNA gene sequencing identification, which belongs to Pseudomonas sp.
The specific process is as follows:
1) Preparation of tea tree anthrax fungus Colletotrichum camelliae fungus cake
Inoculating inclined activated tea tree anthracnose Colletotrichum camellia on PDA flat-plate culture medium, culturing in a constant-temperature incubator at 25deg.C for 7d, and perforating with 7mm aseptic puncher to obtain bacterial cake.
2) Preparation of Pseudomonas palustris Long Nishi strain Pseudomonas palleroniana ZJUCS1001 and Pseudomonas sp
The cryogenically stored Pseudomonas palleroniana ZJUCS strain 1001 strain and the Pseudomonas sp control strain were removed and spread on NA medium plates for 24h (incubation temperature 25 ℃). Single colonies were picked and streaked on NA medium plates for 24h. Then, single colony is picked and inoculated into NA liquid culture medium, and the strain fermentation liquor is obtained by shaking culture for 24 hours at 25 ℃ and 150 rpm.
3) Plate confrontation test
The tea tree anthrax fungus cake is placed in the center of WA flat-plate culture medium, wherein the WA culture medium comprises 5g peptone, 10g glucose, 3g beef extract, 5g sodium chloride and 15g agar powder, the volume is fixed to 1L, and the pH is regulated to 7.2. Then placing aseptic filter paper sheets at 25mm positions on two sides of the fungus cake, taking 10 mu L of fermentation liquor of Pseudomonas palleroniana ZJUCS strain and Pseudomonas sp.
As can be seen from Table 1, the Pseudomonas palleroniana ZJUCS strain 1001 and the Pseudomonas sp control strain have a remarkable inhibition effect on the growth of anthrax fungus hyphae, wherein the inhibition rate of the Pseudomonas palleroniana ZJUCS strain 1001 reaches 71.31%, and the inhibition rate of the Pseudomonas sp control strain is only 46.74%.
Table 1 inhibition of anthrax fungi by ZJUCS1001 Strain
The above experiment was repeated three times to obtain experimental results of the Pseudomonas palleroniana ZJUCS1001 strain and the Pseudomonas sp. Control strain against the plates of anthrax fungus (fig. 1), and the results show that the Pseudomonas palleroniana ZJUCS1001 strain has a remarkable inhibitory effect on the growth of the anthrax fungus hyphae and is remarkably superior to the Pseudomonas sp. Control strain.
Example 4: paylor Long Nishi pseudomonas Pseudomonas palleroniana ZJUCS1001 strain for preventing and controlling anthracnose disease of tea leaves
1) Preparation of tea plant anthrax fungus Colletotrichum camelliae conidium suspension
The tea tree anthrax Colletotrichum camellia after the slant activation is inoculated on a PDA flat-plate culture medium and is placed in a constant-temperature incubator to be cultured for 30 days at 25 ℃. Adding 10ml of sterile water into PDA plate, scraping hypha and conidium with sterile applicator, filtering with 3 layers of sterile mirror paper to obtain conidium suspension, microscopic examining its concentration, adding 0.1% (v/v) Tween-80, and concocting its concentration to 1×10 5 individual/mL of conidium suspension.
2) Preparation of Pseudomonas palustris Long Nishi strain Pseudomonas palleroniana ZJUCS1001 and Pseudomonas sp
The cryogenically stored Pseudomonas palleroniana ZJUCS strain 1001 strain was removed from the control strain and spread on NA medium plates for 24h. Single colonies were picked and streaked on NA medium plates for 24h. Then picking single colony and inoculating into NA liquid culture medium, shake culturing at 25deg.C and 150rpm for 24 hr to obtain strain fermentation broth, and preparing into 1×10 concentration 8 cfu/mL was used for inoculation.
3) Pseudomonas aeruginosa Pseudomonas palleroniana ZJUCS1001 strain and Pseudomonas sp control strain for reducing control effect of anthracnose of tea leaves
Picking young leaves of the 43 varieties of healthy two-year-old Longjing tea trees with similar size and tenderness, cutting Miao Xinsheng, soaking in a 1% sodium hypochlorite solution for 4min, and cleaning with sterile water for surface sterilization. Spraying Pseudomonas palleroniana ZJUCS strain and Pseudomonas sp on the leaf by using an atomizer after the leaf is air-dried until no obvious water stain is on the surfaceControl Strain 1X 10 8 cfu/mL fermentation broth, sterile water as control, is sprayed until the broth drops; the leaves were placed in a square petri dish. After 6h of standing at 25 ℃ in dark, 12 holes are respectively pricked on two sides of the middle veins of the leaf blades by using a sterile needle head, and the conidium liquid of the anthrax fungus is sprayed on the leaf blades by using an atomizer. The leaves are placed in a climatic chamber, the photoperiod of the climatic chamber is set to be 14h/10h after 24h under the dark condition at 25 ℃, and the illumination intensity is 10000lx. After 7d co-cultivation, leaves were taken out for photographing, leaf spot areas were calculated using ImageJ software, and leaf anthracnose morbidity was calculated for tea leaves.
As can be seen from Table 2, both the Pseudomonas palleroniana ZJUCS1001 strain and the Pseudomonas sp control strain have remarkable control effects on anthracnose of tea leaves, wherein the anthracnose incidence of the control group is 56.47%, the anthracnose incidence of tea leaves after Pseudomonas palleroniana ZJUCS1001 inoculation is only 8.39%, the anthracnose infection rate of tea leaves is effectively reduced by 48.08%, and the anthracnose incidence rate of tea leaves after Pseudomonas sp inoculation is 31.59%, and the anthracnose infection rate of tea leaves is only reduced by 24.88%.
Table 2ZJUCS 1001 Strain prevents and treats anthracnose of tea leaves
Fig. 2 and 3 show that both the Pseudomonas palleroniana ZJUCS strain 1001 and the Pseudomonas sp. Control strain have good control effect on anthracnose of tea leaves, wherein the control effect of the Pseudomonas palleroniana ZJUCS strain 1001 reaches 85.14%, and the control effect of the Pseudomonas sp. Strain reaches 44.1%. Therefore, the endophytic bacteria of the Pseudomonas has better control effect on tea tree anthracnose, in particular to Pseudomonas palleroniana ZJUCS strain 1001, and the control effect is obviously better than that of a Pseudomonas sp.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (3)
1. Pseudomonas paleholder Long Nishi (Pseudomonas palleroniana) strain ZJUCS1001, characterized by: the preservation number is CGMCC NO:24722.
2. use of pseudomonas paler Long Nishi strain ZJUCS1001 according to claim 1, wherein: inhibiting anthracnose of tea trees;
the tea tree anthracnose inhibition is to inhibit the growth of tea tree anthracnose pathogenic bacteria camellia anthracnose (Colletotrichum camelliae).
3. Use of a pseudomonas palettes Long Nishi strain according to claim 2, characterized in that: the inhibiting of the growth of the tea tree anthrax pathogenic bacteria comprises inhibiting the growth of pathogenic bacteria hyphae.
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