CN117701430A - Pseudomonas friedrisburgh and application thereof in preventing and controlling plant diseases - Google Patents
Pseudomonas friedrisburgh and application thereof in preventing and controlling plant diseases Download PDFInfo
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- CN117701430A CN117701430A CN202311692730.8A CN202311692730A CN117701430A CN 117701430 A CN117701430 A CN 117701430A CN 202311692730 A CN202311692730 A CN 202311692730A CN 117701430 A CN117701430 A CN 117701430A
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Abstract
The invention discloses pseudomonas friedrisburghii and application thereof in preventing and treating plant diseases. The invention discloses a Pseudomonas friedreich which is Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33 and has a preservation number of CGMCC No.28557 in the China general microbiological culture Collection center. Experiments prove that the friedrenches pseudomonas used for soaking seeds and root irrigation have obvious control effects on plant diseases, and the friedrenches pseudomonas used for soaking seeds and root irrigation have good application prospects.
Description
Technical Field
The invention relates to pseudomonas friedrisburgh in the field of plant protection and application thereof in preventing and controlling plant diseases.
Background
Pseudomonas bacteria are typical rhizosphere growth promoting bacteria, and not only have the capability of promoting growth and directly antagonizing pathogenic bacteria, but also some extracellular substances produced by the Pseudomonas, such as extracellular polysaccharide, protein, secondary metabolites and the like, can induce plants to generate a series of reactions, so that the disease resistance of the plants is enhanced. These reactions include activating the immune system of the plant, enhancing the stability of the plant cell wall, promoting plant growth and development, and the like. However, there has been no report on the study of pseudomonas friedrikombu in inducing disease resistance in plants. Based on the background, the screening of the pseudomonas friedri with the biocontrol potential and obvious disease resistance effect on plant diseases can provide novel biocontrol resources for the control of the plant diseases.
Disclosure of Invention
The invention aims to solve the technical problem of how to control plant diseases.
In order to solve the technical problems, the invention firstly provides Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33 which has a preservation number of CGMCC No.28557 in the China general microbiological culture Collection center.
The invention also provides a microbial inoculum, and the active ingredient of the microbial inoculum is the Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33.
The microbial inoculum can be used for preventing and treating plant diseases or inhibiting the growth of plant pathogenic microorganisms.
In the microbial inoculum, the inorganic phosphorus can be at least one of calcium hydrophosphate, phosphate rock powder, ferric phosphate, aluminum phosphate and calcium phosphate; the organophosphorus may be lecithin.
In the microbial inoculum, the microbial inoculum can also comprise a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be mineral material, plant material or 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, soy flour and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of living cells being cultured, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and filtrate. The formulation of the composition can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.
Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.
The invention also provides the application of the pseudomonas friedrisburgh (Pseudomonas frederiksbergensis) YK33 or any one of the following microbial inoculum:
1) Preventing and treating plant diseases;
2) Preparing a product for controlling plant diseases;
3) Inhibiting the growth of phytopathogenic microorganisms;
4) Preparation of a composition for inhibiting growth of phytopathogenic microorganisms.
In the above application, the disease may be a disease caused by a plant pathogenic bacterium or a pathogenic fungus; the phytopathogenic microorganism may be a phytopathogenic bacterium or a pathogenic fungus.
In the above application, the pathogenic bacteria may be Pseudomonas amygdalina lacrimation pathogenic variety (Pseudomonas amygdali pv.lachryrns), citrullus acidovorax (Acidovorax citrulli), or Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae pv.tomo);
the pathogenic fungus may be watermelon dichotoma (Ascochyta citrullina), rhizoctonia solani (Rhizoctonia solani), alternaria solani (Alternaria solani), phytophthora solani (Stemphylium solani), or Phytophthora capsici (Phytophthora capsica Leonian).
In the above application, the plant may be M1) or M2) or M3):
m1) dicotyledonous or monocotyledonous plants;
m2) cucurbitaceae or solanaceae;
m3) cucumber or tomato.
The present invention also provides a method of controlling plant diseases, the method comprising: the friedrix pseudomonas (Pseudomonas frederiksbergensis) YK33 or the microbial inoculum is applied to plants to realize the control of plant diseases.
In the above method, applying the pseudomonas fredrikohlrabi (Pseudomonas frederiksbergensis) YK33 or the microbial agent to the plant comprises soaking seeds of the plant or root irrigation of the plant.
In the above method, the disease may be a disease caused by a plant pathogenic bacterium or a pathogenic fungus.
Further, the pathogenic bacteria may be Pseudomonas amygdalina lacrimation pathogenic varieties (Pseudomonas amygdali pv.lachryrns), watermelon acidovorax (Acidovorax citrulli), or Pseudomonas syringae tomato pathogenic varieties (Pseudomonas syringae pv.118);
the pathogenic fungus may be watermelon dichotoma (Ascochyta citrullina), rhizoctonia solani (Rhizoctonia solani), alternaria solani (Alternaria solani), phytophthora solani (Stemphylium solani), or Phytophthora capsici (Phytophthora capsica Leonian).
In the above method, the plant may be M1) or M2) or M3):
m1) dicotyledonous or monocotyledonous plants;
m2) cucurbitaceae or solanaceae;
m3) cucumber or tomato.
In one embodiment of the invention, the plant disease is melon fruit blotch. In another embodiment of the present invention, the plant disease is cucumber angular leaf spot.
The friedrix burg pseudomonas (Pseudomonas frederiksbergensis) YK33 seed soaking and root irrigation treatments of the invention have remarkable disease-resistant induction effects on plant diseases, the control effects on melon fruit blotches are respectively 62.67% and 64.69%, and the control effects on cucumber angular blotches are respectively 66.71% and 65.73%. In conclusion, the friedel-crafts pseudomonas (Pseudomonas frederiksbergensis) YK33 is the friedel-crafts pseudomonas with potential biocontrol application potential for inducing plant disease resistance, is comprehensively reported from a seed treatment and root irrigation treatment for the first time, has obvious induction effect on plant bacterial diseases of melon fruit blotch and cucumber angular blotch, and has good application prospect.
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
Description of biological Material preservation
The preservation unit is abbreviated as: CGMCC
Preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Deposit unit address: beijing, chaoyang, north Chen West Lu 1, 3, postal code: 100101
Preservation date: 2023 9 month 26 day
Accession numbers of the preservation center: CGMCC No.28557
Classification naming: pseudomonas friedrikosburgh (Pseudomonas frederiksbergensis)
Strain number: YK33
Drawings
FIG. 1 shows colony morphology (A) of strain YK33 and colony morphology (B) after gram staining.
FIG. 2 is a polygenic phylogenetic tree of strain YK33.
FIG. 3 is a relative expression analysis of marker genes and related plant disease resistance genes for different hormone pathways in melon seed embryos by different strain seed soaking treatments. And (3) injection: * P <0.01;0< Log2<1: up-regulating expression; log2>1, significantly up-regulated expression.
FIG. 4 is an analysis of the antagonistic capacity of Pseudomonas friedreich by YK33 against different bacterial and fungal pathogens. The pseudomonas syringae tomato leaf spot disease pathotype is pseudomonas syringae tomato pathotype (Pseudomonas syringae pv. Phytophthora is Phytophthora capsici (Phytophthora capsica Leonian).
FIG. 5 shows the control effect of the seed soaking induction treatment of Pseudomonas friedreggy 33 on melon fruit blotch.
FIG. 6 shows the control effect of P.friedrenches YK33 root-irrigation induction treatment on melon fruit blotches.
FIG. 7 shows the control effect of Pseudomonas friedreggii YK33 seed soaking induction treatment on cucumber angular leaf spot.
FIG. 8 shows the control effect of Pseudomonas friedreich by YK33 root irrigation induction treatment on cucumber angular leaf spot.
Detailed Description
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were each set up for at least three replicates and the results averaged.
Watermelon fruit two spore (Ascochyta citrullina) in the following examples has been described: zhao Yanjie, li Baoju, dan Yanxia. Occurrence of melon gummy stem blight, prevention and cure [ J ]. Chinese vegetables, 2008 (02): 56-57+70. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
Rhizoctonia solani (Rhizoctonia solani) in the following examples has been described: li Lei, chen Lida, huang Yishuo, xie Xuewen, dan Yanxia, chai Ali, li Baoju. Set up and use of real-time fluorescent quantitative PCR detection System for potato black nevus bacteria. Agricultural Biotechnology journal, 2021,29 (07): 1417-1425. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
Alternaria solani (Alternaria solani) in the following examples is described: guo Runting, dan Yanxia, zhao Qian, etc. Alternaria lettuce leaf spot pathogen identification [ J ]. Plant pathogenicity report, 2018,48 (03): 418-422. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
The calicheapest (Stemphylium solani) in the following examples is described in: xie Xuewen, chen Lida, cao Jinjiang, han Daojie, dan Yanxia, chai Ali, li Lei, li Baoju. Methods for real-time fluorescent quantitative PCR detection of Rhizopus solani were established and applied. Plant pathology report, 2021,51 (04): 618-625.DOI:10.13926/j. Cnki. Apps.000717. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
Phytophthora capsici (Phytophthora capsica Leonian) in the following examples has been described: jiang Houchun Lv Guo Hua, dan Yanxia. Phytophthora capsici [ J ] of capsicum seed is detected by selective medium, 2011 (06): 58-61. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
The pathogenic variant of the tearing of Pseudomonas amygdalina (Pseudomonas amygdali pv. Lachryrnans) in the examples below has been described: li Lei pathogenic bacteria of bacterial gummosis of cucumber based on whole genome sequencing and comparative genome analysis [ D ]. National academy of agricultural sciences, 2019. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
The watermelon acidovorax (Acidovorax citrulli) in the following examples is the strain of watermelon acidovorax Aac5, which is disclosed in the documents "Tian Hui, xie Xuewen, li Huanling, wei Songgong, li Baoju. Li Baoju, doctor diagnosis and hand (seventy) stock pumpkin seed-borne bacterial fruit blotch, and the prevention and treatment of the disease, chinese vegetables 2014, (05): 65-66", which are available to the public from the national academy of agricultural sciences vegetable flowers research to repeat the present application. And cannot be used for other purposes.
The pathogenic variety of Pseudomonas syringae (Pseudomonas syringae pv.total) in the following examples has been described: chai Ali, pati Guli, guo Weitao, dan Yanxia, xie Xuewen, xi Xianmei, li Baoju. Establishment and application of a real-time fluorescent quantitative PCR detection method for tomato bacterial Spot pathogens. Gardening journal, 2019,46 (01): 182-192. The public is available from vegetable and flower research institute of national academy of agricultural sciences. The biomaterial was used only for repeated experiments of the invention and was not used for other purposes.
The melon in the following example is sheep horn honey (variety registration number: GPD melon (2017) 110042), beijing Co., ltd.
The cucumber in the following example is "zhongnong No. 6", beijing limited, a science and technology of the medium vegetable seed industry.
EXAMPLE 1 isolation and screening of high-efficient induced-resistance Strain
The inventors isolated 138 bacterial strains from the field rhizosphere soil of harbine. The separation method comprises the following steps:
1. isolation of strains
10g of rhizosphere soil is weighed and dissolved in 90mL of sterile water, and the shaking table is used for shaking treatment for 30min at the constant temperature of 28 ℃ to ensure that the rhizosphere soil is fully and uniformly mixed.Mixing the soil sample according to 10 -1 To 10 -5 Performing gradient dilution, and respectively sucking 100 μl of diluted solution with concentration of 10 -3 、10 -4 、10 -5 Coating NA flat plate (peptone 10.0, beef powder 3.0g/L, sodium chloride 5.0g/L, agar powder 15.0g/L, pH value 7.2+ -0.2), coating 3 flat plates each, culturing at 28 deg.C constant temperature for 24h, picking single colony with different forms, streaking and purifying on sterile NA flat plate until single colony with the same form is grown, picking single colony, and culturing in NA liquid culture medium.
2. Melon seed germination induction
Diluting the bacterial suspension cultivated in the step 1 by 100 times, adding 10mL into a culture dish containing filter paper sheets, placing 30 melon seeds into a culture box, accelerating germination at 28 ℃ for 36h, and counting the germination rate to ensure that the germination induction treatment condition cannot influence the germination of the melon seeds. The budding rate calculation formula is: the germination rate= (number of germinated melon seeds/total melon seeds) ×100%.
3. Melon seed sowing
Planting melon seeds subjected to seed soaking and germination accelerating of different bacteria in a 72-hole seedling pot for cultivation in a greenhouse, and taking the melon seedlings as a treatment group to prepare for inoculation of pathogenic bacteria when the melon seedlings grow to two cotyledons. And setting healthy control (germination accelerating according to step 2 by using clear water without pathogen) and clear water control (germination accelerating according to step 2 by using clear water, pathogen inoculation).
4. Preparation of pathogenic bacteria suspension
When the melon seedlings after induction grow to 2 cotyledons, the stored test strain, namely the watermelon acidovorax Aac5 strain, is taken out from a refrigerator at the temperature of minus 80 ℃. Taking out, melting in ice bath, activating on NB plate at 28deg.C, transferring to NB liquid culture medium 200 r.min -1 Culturing to obtain pathogenic bacteria suspension with concentration of about 10 6 cfu/mL。
5. Inoculation of pathogenic bacteria
The cultured concentration is 10 6 cfu/mL of Aac5 bacterial suspension was used to inoculate two cotyledon stage melon with a micro-sprayer. Respectively placing in a moisturizing cabinet, maintaining the temperature at about 20deg.C and relative humidity at 90% RH, observing when melon plants are illThe occurrence of an indoor healthy control records the disease index.
6. Disease investigation
Observation is carried out every day after inoculation, the disease condition is recorded, the disease level is determined, and the disease index and the prevention effect are calculated. Leaf disease severity grading criteria (i.e., disease grading criteria): each plant was examined for two cotyledons and rated as a percentage of the lesion area of each leaf over the entire leaf area.
Disease grading standard:
level 0: no disease spots;
stage 1: the disease spots occupy less than 5 percent (including 5 percent) of the leaf area;
3 stages: 5% -25% of the leaf area of the disease spots (excluding 5% and 25%);
5 stages: the disease spots account for 25% -50% of the leaf area; (25% not included, 50% included)
7 stages: 50% -75% of the leaf area of the disease spots (excluding 50% and 75%);
stage 9: the lesions occupy more than 75% of the leaf area (excluding 25%).
And (3) data processing: the disease index of melon leaves treated with each strain was calculated using Excel 2010.
Control (%) = ((fresh water control group disease index-treatment group disease index)/fresh water control group disease index) ×100%.
7. Test results
(1) Living screening
By the way of seed soaking and melon seed treatment, the strain YK33 with the highest control effect on watermelon acidovorax is selected from 138 strains, and the control effect is 61.7+/-1.6%.
EXAMPLE 2 identification and preservation of YK33 Strain
(1) Morphological observation
After the strain YK33 having the best control effect selected in example 1 was cultured on a plate of a medium (peptone 10.0, beef powder 3.0g/L, sodium chloride 5.0g/L, agar powder 15.0g/L, pH 7.2.+ -. 0.2) for 72 hours, the colony morphology was observed. As shown in FIG. 1, the colony is round, convex, opaque, moist and smooth in surface, regular in edge, light yellow and free of spores.
(2) Physiological and biochemical assays
By referring to methods of "Berger's Manual of bacteria identification" and "Manual of common bacterial System identification", strain YK33 was subjected to physiological and biochemical tests such as gram stain, growth pH test, salt tolerance test, motility test, contact enzyme test, V-P test, starch hydrolysis test, gelatin liquefaction test, citrate utilization test, nitrate reduction test, etc. The medium formulation used was as follows: 10.0 g/L peptone, 3.0g/L beef powder, 5.0g/L sodium chloride, 15.0g/L agar powder and pH value of 7.2+/-0.2.
The results are shown in Table 1. The results showed that strain YK33 is a gram negative bacterium (fig. 1), tolerant to acidic ph=6, poorly tolerant to salt ions (mass percent), and mobile. The contact enzyme, V-P and gelatin liquefaction reactions were positive, and citrate utilization, starch hydrolysis and nitrate reduction reactions were negative.
TABLE 1 physiological biochemical reaction measurement results of strain YK33
Note that: + indicates that the test result is positive; -indicating that the experimental result is negative. A negative in the pH and salt tolerance test indicates that the strain is not growing, and a positive indicates that the strain is growing.
(3) Biolog assay
Single colony of the strain YK33 is selected and inoculated on the inclined surface of a test tube of the NB medium, and the strain YK33 is cultured for 24 hours at the temperature of 28 ℃. The unique carbon source utilization assay for strain YK33 was performed by the China center for type culture Collection of microorganisms using a BIOLOG GENIII kit (according to the kit instructions).
As a result, it was revealed that the YK33 strain can use glucose, D-fructose, D-trehalose, L-trehalose, citric acid, etc., but cannot use glucan, D-gentiobiose, sucrose, D-sorbitol, etc., table 2.
TABLE 2 BIOLOG assay results for Strain YK33
Note that: + indicates that the test result is positive; -indicating that the experimental result is negative.
(4) Multi-gene phylogenetic tree construction
After genomic DNA of strain YK33 was extracted using a general bacterial genomic extraction kit, the sequences were amplified by PCR using bacterial 16s DNA, gyrB and rpoD genes, respectively (the primers used are shown in Table 3), and the obtained PCR amplified products were sent to Bomaide biosystems for sequencing. And (3) constructing a phylogenetic tree by using a maximum likelihood method after MEGA6.0 comparison, determining the classification status of the strain YK33, and analyzing the genetic relationship. The 16s DNA, gyrB and rpoD gene sequences of the strain YK33 are respectively shown as SEQ ID No.1-SEQ ID No.3 in the sequence table.
A polygenic phylogenetic tree of strain YK33 (FIG. 2) was constructed based on the 16s DNA, gyrB and rpoD genes and identified as Pseudomonas friedreich (Pseudomonas frederiksbergensis), which was designated as Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33.
TABLE 3 primer Gene sequences required for polygene identification
Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33 was deposited at the China general microbiological culture Collection center (CGMCC; address: north Chen West Lu No.1, 3, institute of microbiology, china academy of sciences; postal code: 100101) at the 9 th month 26 year 2023, with a deposit number of CGMCC No.28557.
EXAMPLE 3 analysis of expression level of plant disease resistance-related Gene in melon seed by YK33 seed soaking treatment
(1) Melon seed induction treatment
Soaking melon seeds in 2% NaClO solution for 10min, washing with sterile water, and air drying. Fermentation broth (OD) using strain YK33 600 =1.0) after 100-fold dilution, seed soaking and germination accelerating melon seeds (see example 1) were performed, and each group was repeated 3 times. Using the FZB42 strain as a control, the Bacillus belicus (Bacillus velezensis) FZB42 strain is described in the literature "Fan B, wang C, song X, ding X, wu L, wu H, gao X and Borriss R (2018) Bacillus velezensis FZB in 2018: the Gram-Positive Model Strain for Plant Growth Promotion and biocontrol. Front. Microbiol.9:2491.Doi: 10.3389/fmib. 2018.02491".
(2) Extraction of plant RNA
After 24 hours of seed soaking, the germinated seed embryos are extracted with plant RNA. Plant tissue was quickly frozen with liquid nitrogen and ground as soon as possible. The ground sample was subjected to RNA extraction and reverse transcription using a plant RNA extraction kit (Tiangen, DP 419), and after the reverse transcription was completed, the sample was diluted to 100 ng/. Mu.L for qPCR.
(3) Transcription analysis of melon-related defensive genes by q-PCR
The gene expression was determined by fluorescent quantitative polymerase chain reaction (qPCR). The primers used are shown in Table 4, 3 replicates were set per sample, sterile water was used as a negative control to replace template DNA, and changes in fluorescent signal were monitored and recorded in real time.
TABLE 4 RT-qPCR primer sequences for plant defense gene analysis
(4) Data analysis
The reference gene ACT is used as a standard to relatively quantify the target gene, and 2 is adopted for calculation -ΔΔCt The method comprises the following steps:
relative expression amount =2 -ΔΔCt ＝2 -[(CtE-CtF)]-[(CtA-CtB)] . Wherein CtA is the Ct value of the gene to be tested before treatment; ctB is the Ct value of the reference gene before treatment; CE is the Ct value of the gene to be measured after treatment; ctF is the Ct value of the reference gene after treatment.
The test results are shown in fig. 3, and the key synthetic genes of salicylic acid and jasmonic acid/ethylene hormone pathways in plant immunity are used as targets, so that the significant up-regulated expression of jasmonic Acid (AOS)/ethylene (EIN 2) pathway marker genes and the significant up-regulated expression of plant disease resistance gene (PR 1) can be activated by the seed soaking treatment of the strain YK33 and FZB42, and the resistance of a plant system can be activated by the seed soaking induction treatment of the strain YK33.
EXAMPLE 4 antagonistic ability of Pseudomonas friedreggii YK33 against common 8 strains of pathogenic bacteria
The antagonistic capacity analysis of 3 pathogenic bacteria and 5 pathogenic fungi is carried out by using a filter paper sheet culture method to detect the Pseudomonas friedrisburghii YK33.
The 5 pathogenic fungi used were: watermelon husk two spores (Ascochyta citrullina), rhizoctonia solani (Rhizoctonia solani), alternaria solani (Alternaria solani), calicheapest (Stemphylium solani), phytophthora capsici (Phytophthora capsica Leonian); the 3 pathogenic bacteria are: pseudomonas amygdalina lacrimation pathogenic variant (Pseudomonas amygdali pv.lachryrns), watermelon acidovor (Acidovorax citrulli), pseudomonas syringae tomato pathogenic variant (Pseudomonas syringae pv.tomo).
The specific method comprises the following steps:
pseudomonas friedrisburgh YK33 suspension: activating the strain YK33 preserved at-80 ℃ on an LB plate by a streaking method, culturing for 1d at 28 ℃, selecting single bacterial colony, transferring into a shaking tube, culturing at 28 ℃ and 180rpm for 24h, and obtaining YK33 bacterial suspension.
Analysis of bacterial antagonistic ability: diluting the bacterial suspension of different pathogenic bacteria cultured for 48 hours to 10 8 CFU/mL, 100. Mu.L was applied to NA plate and then allowed to air dry. A filter paper sheet was attached to the middle of the plate, and 5. Mu.L of a suspension of Pseudomonas friedrisburghii YK33 (1X 10) 8 CFU/mL) was added to a filter paper sheet and incubated at 28 degrees CelsiusAfter 48 hours of culture in the box, the diameter of the inhibition zone is measured, and the inhibition rate is calculated. As a control, 5. Mu.L of sterile water was used. Each treatment was repeated 3 times. Antibacterial ratio (%) =antibacterial diameter/control colony diameter×100. Culture medium for pathogenic bacteria: NB medium (beef extract 3g, peptone 5g, glucose 2.5g, distilled water 1000ml, pH 7.0).
Analysis of fungal antagonistic ability: inoculating pathogenic fungi cake with diameter of 8mm (the pathogenic fungi cake with diameter of 8mm cut after culturing on PDA plate) at center of PDA plate of experimental group, culturing at 28deg.C for 24 hr, respectively dripping 5 μl of Morganella virens YK33 suspension (1×10) at 4 points 3cm away from center of plate by crisscross method 8 CFU/mL), 5d at 28 ℃, 3 replicates of 3 plates per treatment. Only the fungal cake was cultured as a control group (CK).
Results As shown in FIG. 4, YK33 has no obvious antagonism to 8 common plant bacterial fungal pathogens, and the prevention mechanism of the screened strain YK33 taking melon fruit blotch as a target under potted plant bioassay is proved from the side to induce plant system resistance.
EXAMPLE 5 determination of the efficacy of seed soaking Induction treatment method of Pseudomonas friedel-crafts YK33 on melon fruit Spot disease
1. Picking single colony of Pseudomonas friedregis YK33 and inoculating to NB medium (peptone 10.0, beef powder)
3.0g/L, 5.0g/L sodium chloride, pH 7.2+ -0.2), shaking culture at 28deg.C to a concentration of 1×10 8 cfu·mL -1 And (5) standby.
2. Picking single colony of watermelon acidovorax Aac5 strain, inoculating into NB medium, shake culturing at 28deg.C to 1×10 8 cfu·mL -1 And (5) standby.
3. Diluting the suspension of the Pseudomonas friedrisburgii YK33 obtained in the step 1 by 100 times, adding 10mL into a culture dish containing filter paper sheets, placing 30 melon seeds into an incubator, and accelerating germination at 28 ℃ for 36h; then planting the germinated seeds in a 72-hole seedling pot for cultivation in a greenhouse; when melon seedlings grow to two cotyledons, inoculating the watermelon acidovorax 5 bacterial suspension obtained in the step 2, placing in a moisturizing cabinet, keeping the temperature at about 26 ℃ and the relative humidity at 90% RH, recording the disease condition after obvious melon and fruit spot symptoms appear on melon plants in a negative control group, determining the disease level, calculating the disease index and the prevention effect, and calculating the disease grading standard and the disease index as in the example 1. A negative control (CK, replacing the vireri pseudomonas forti YK33 bacterial suspension with sterile water) and a reagent control (replacing the vireri pseudomonas forti YK33 bacterial suspension with a fluxazole activated ester liquid (solvent is water, solute is fluxazole activated ester (FBT), concentration of fluxazole activated ester is 20 mg/L) were set. The experiment was repeated 3 times to measure the results.
Control (%) = ((negative control group disease index-treatment group disease index)/negative control group disease index) ×100%.
4. Results
As shown in fig. 5, the disease index (25.13±1.1) of the YK33 treatment group and the disease index (34.55±2.6) of the drug control group were significantly lower than those of the negative control group (67.31±3.2); the melon induced by the YK33 bacterial suspension has a control effect on watermelon acidovorax Aac5 up to 62.67%, which is obviously higher than that of the control medicament epoxiconazole activated ester (48.67%), which indicates that the Frederick burger pseudomonas YK33 has a good control effect on watermelon acidovorax Aac 5.
TABLE 5 control Effect of Pseudomonas friedreich YK33 on watermelon acidovorax Aac5
Example 6 determination of the efficacy of treatment method of root-irrigation induction of Pseudomonas friedrenches YK33 on melon fruit Spot disease
1. Picking single colony of Pseudomonas friedrisburghii YK33, inoculating into NB medium, shake culturing at 28deg.C to 1×10 8 cfu·mL -1 And (5) standby.
2. Picking single colony of melon fruit spot disease germ (watermelon acidovorax Aac5 strain) and inoculating into NB culture medium, shake culturing at 28deg.C to 1×10 8 cfu·mL -1 And (5) standby.
3. Inoculating: melon seeds are planted in a 72-hole seedling pot and are cultivated in a greenhouse; and (3) when melon seedlings grow to a true leaf stage, root irrigation induction is carried out by using the Pseudomonas fries YK33 bacterial suspension obtained in the step (1), 50mL of each cucumber seedling is quantitatively irrigated, and the induction is carried out 1 time every five days, and the total induction is carried out 3 times. And 3 days after the 3 rd induction is finished, the melon fruit spot disease germ suspension obtained in the step 2 is inoculated by adopting a spraying method, and moisture is preserved after inoculation.
After the negative control melon plants have obvious melon and fruit spot symptoms, the disease conditions are recorded, the disease grade is determined, the disease index and the prevention effect are calculated, and the disease grading standard and the disease index are calculated as in example 1. A negative control (CK, replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with sterile water) and a reagent control (replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with a fluxazole activated ester liquid medicine (solvent is water, solute is fluxazole activated ester, and concentration of the fluxazole activated ester is 20 mg/L)) were set. The experiment was repeated 3 times to measure the results.
Control (%) = ((negative control group disease index-treatment group disease index)/negative control group disease index) ×100%.
4. Results
As shown in fig. 6, the disease index (21.26±1.76) of the YK33 treatment group and the disease index (28.92 ±1.43) of the drug control group were significantly lower than those of the negative control group (60.21±1.33); the melon induced by the YK33 bacterial suspension root irrigation method has a control effect of 64.69 percent on melon fruit blotch pathogens, which is obviously higher than that of the control medicament epoxiconazole activated ester (51.97 percent), thus showing that the Frederickburg pseudomonas YK33 has a good control effect on melon fruit blotch pathogens.
TABLE 6 control effect of Pseudomonas friedregger YK33 on melon fruit blotch
EXAMPLE 7 determination of the control Effect of Pseudomonas friedrenching treatment method on cucumber angular leaf spot by means of Pseudomonas friedrenches YK33
1. Picking single colony of Pseudomonas friedrisburghii YK33, inoculating into NB medium, shake culturing at 28deg.C to 1×10 8 cfu·mL -1 And (5) standby.
2. Picking single colony of cucumber angular leaf spot pathogen Pal-nm002 (Pseudomonas amygdalina lacrimation pathogenic variety (Pseudomonas amygdali pv. Lachryrnans)) and inoculating into NB medium, shake culturing at 28deg.C to 1×10 concentration 8 cfu·mL -1 And (5) standby.
3. Diluting the suspension of the Pseudomonas friedrisburgii YK33 obtained in the step 1 by 100 times, adding 10mL into a culture dish containing filter paper sheets, placing 30 cucumber seeds into an incubator, and accelerating germination at 28 ℃ for 36h; then planting the germinated seeds in a 72-hole seedling pot for cultivation in a greenhouse; and (3) inoculating the cucumber angular leaf spot disease germ suspension obtained in the step (2) to the cucumber after the cucumber seedlings grow to two leaves and one heart period, placing the cucumber angular leaf spot disease germ suspension in a moisturizing cabinet, keeping the temperature at about 26 ℃ and the relative humidity at 90% RH, recording the disease condition after the cucumber in the negative control group has obvious angular leaf spot symptoms, determining the disease level, calculating the disease index and the prevention effect, and calculating the disease grading standard and the disease index as in the embodiment 1. A negative control (CK, replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with sterile water) and a reagent control (replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with a fluxazole activated ester liquid medicine (solvent is water, solute is fluxazole activated ester, and concentration of the fluxazole activated ester is 20 mg/L)) were set. The experiment was repeated 3 times to measure the results.
Control (%) = ((negative control group disease index-treatment group disease index)/negative control group disease index) ×100.
4. Results
As shown in fig. 7, the disease index (26.21±1.1) of the YK33 treatment group and the disease index (35.55±2.6) of the drug control group were significantly lower than those of the negative control group (78.73±6.2); the prevention effect of the cucumber subjected to induction treatment by adopting the YK33 bacterial suspension seed soaking mode on the angular leaf spot of the cucumber is up to 66.71 percent, which is obviously higher than the prevention effect (54.85 percent) of the control medicament fluxazole activated ester, which indicates that the Friedel-crafts Pseudomonas pseudomonad YK33 has good prevention and treatment effect on the angular leaf spot of the cucumber.
TABLE 7 control effect of Pseudomonas friedreich YK33 on cucumber angular leaf spot
Example 8 determination of the control Effect of Pseudomonas friedrenches YK33 root-irrigation Induction treatment method on cucumber angular leaf Patches
1. Picking single colony of Pseudomonas friedrisburghii YK33, inoculating into NB medium, shake culturing at 28deg.C to 1×10 8 cfu·mL -1 And (5) standby.
2. Picking single colony of cucumber angular leaf spot pathogen Pal-nm002 (Pseudomonas amygdalina lacrimation pathogenic variety (Pseudomonas amygdali pv. Lachryrnans)) and inoculating into NB medium, shake culturing at 28deg.C to 1×10 concentration 8 cfu·mL -1 And (5) standby.
3. Inoculating: planting cucumber seeds in a 72-hole seedling pot for cultivation in a greenhouse; and (3) when cucumber seedlings grow to a true leaf stage, root irrigation induction is carried out by using the pseudomonas friedel YK33 bacterial suspension obtained in the step (1), the root irrigation of each cucumber seedling is 50mL, the induction is carried out 1 time every five days, and the total induction is carried out 3 times. And 3 days after the 3 rd induction is finished, the cucumber angular leaf spot bacteria suspension obtained in the step 2 is inoculated by adopting a spraying method, and moisture is preserved after inoculation.
After the cucumber in the negative control group has obvious angular spot symptoms, the disease condition is recorded, the disease grade is determined, the disease index and the prevention effect are calculated, and the disease grading standard and the disease index are calculated as in example 1. A negative control (CK, replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with sterile water) and a reagent control (replacing the fredribbe pseudomonas fredribbe YK33 bacterial suspension with a fluxazole activated ester liquid medicine (solvent is water, solute is fluxazole activated ester, and concentration of the fluxazole activated ester is 20 mg/L)) were set. The experiment was repeated 3 times to measure the results.
Control (%) = ((negative control group disease index-treatment group disease index)/negative control group disease index) ×100%.
4. Results
As shown in fig. 8, the disease index (20.31±1.7) of the YK33 treatment group and the disease index (32.14±3.4) of the drug control group were significantly lower than those of the negative control group (59.27 ±1.3); the prevention effect of the cucumber subjected to induction treatment by adopting a YK33 bacterial suspension root irrigation mode on the angular leaf spot of the cucumber is up to 65.73 percent, which is obviously higher than the prevention effect (45.77 percent) of the control medicament fluxazole activated ester, which indicates that the Friedel-crafts Pseudomonas pseudomonad YK33 has good prevention and treatment effect on the angular leaf spot of the cucumber.
TABLE 8 control effect of Pseudomonas friedreich YK33 on cucumber angular leaf spot
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. Pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33 with a preservation number of CGMCC No.28557 in China general microbiological culture Collection center.
2. A microbial inoculum, characterized in that: the active ingredient of the microbial inoculum is pseudomonas friedrisburghii (Pseudomonas frederiksbergensis) YK33 as claimed in claim 1.
3. Use of pseudomonas friedrisburgh (Pseudomonas frederiksbergensis) YK33 according to claim 1 or any one of the following bacterial agents according to claim 2:
1) Preventing and treating plant diseases;
2) Preparing a product for controlling plant diseases;
3) Inhibiting the growth of phytopathogenic microorganisms;
4) Preparation of a composition for inhibiting growth of phytopathogenic microorganisms.
4. A use according to claim 3, characterized in that: the disease is caused by plant pathogenic bacteria or pathogenic fungi; the plant pathogenic microorganism is plant pathogenic bacteria or pathogenic fungi.
5. The use according to claim 4, characterized in that: the pathogenic bacteria are Pseudomonas amygdalina lacrimation pathogenic varieties (Pseudomonas amygdali pv.lachryrns), watermelon acidovorax (Acidovorax citrulli), or Pseudomonas syringae tomato pathogenic varieties (Pseudomonas syringae pv.tomo);
the pathogenic fungi are watermelon dichotoma (Ascochyta citrullina), rhizoctonia solani (Rhizoctonia solani), alternaria solani (Alternaria solani), calicheapest (Stemphylium solani), or phytophthora capsici (Phytophthora capsica Leonian).
6. Use according to any one of claims 3-5, characterized in that: the plant is M1) or M2) or M3):
m1) dicotyledonous or monocotyledonous plants;
m2) cucurbitaceae or solanaceae;
m3) cucumber or tomato.
7. A method of controlling plant diseases comprising: the control of plant diseases is achieved by applying the pseudomonas friedrisburgh (Pseudomonas frederiksbergensis) YK33 according to claim 1 or the microbial inoculum according to claim 2 to plants.
8. The method according to claim 7, wherein: applying the pseudomonas friedreich (Pseudomonas frederiksbergensis) YK33 or the microbial agent to a plant includes soaking seeds of the plant or root-filling the plant.
9. The method according to claim 7 or 8, characterized in that: the disease is caused by plant pathogenic bacteria or pathogenic fungi;
further, the pathogenic bacteria are Pseudomonas amygdalina lacrimation pathogenic varieties (Pseudomonas amygdali pv.lachryrns), watermelon acidovorax (Acidovorax citrulli), or Pseudomonas syringae tomato pathogenic varieties (Pseudomonas syringae pv.118);
the pathogenic fungi are watermelon dichotoma (Ascochyta citrullina), rhizoctonia solani (Rhizoctonia solani), alternaria solani (Alternaria solani), calicheapest (Stemphylium solani), or phytophthora capsici (Phytophthora capsica Leonian).
10. The method according to any one of claims 7-9, characterized in that: the plant is M1) or M2) or M3):
m1) dicotyledonous or monocotyledonous plants;
m2) cucurbitaceae or solanaceae;
m3) cucumber or tomato.
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