CN114032182B - Fungus with functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth - Google Patents

Abstract

The invention discloses a fungus with the functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth. The invention provides Talaromyces liani, the strain number of which is DS59-19F, and the registration number of which in China general microbiological culture Collection center (CGMCC) is 23222. The strain can simultaneously antagonize a plurality of garlic root rot pathogens, including garlic root rot pathogens which are first discovered in China, namely, horn fungus (Ceratobasidium sp.) and acanthocellulose terrestris (Setophoma terrestris). In addition, the strain also has obvious plant growth promoting function. The strain has wide application prospect in preventing and treating garlic root rot and promoting garlic sprout growth so as to improve garlic yield.

Description

Fungus with functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth

Technical Field

The invention relates to the field of biological pesticides, in particular to a fungus with the functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth.

Background

Garlic is a perennial herb of Allium genus of Liliaceae family, is one of the aromatic vegetables favored by people all over the country, and is also an important cash crop in China. With the increasing demand of garlic, the planting area and continuous planting period of garlic are continuously increased, so that continuous cropping obstacle is also called Soil decay, which is shown as outbreak of garlic root rot, and a plurality of serious problems are brought to agricultural production, ecological environment and food safety. Researches show that the pathogenic bacteria of garlic root rot in China mainly comprise a plurality of pathogenic bacteria such as fusarium oxysporum (Fusarium oxysporum), fusarium layering (Fusarium proliferatum), fusarium fingium (Fusarium redolens), pythium (Phthium) and the like; it has also been found that the continuous planting of garlic or other crops of the allium genus causes accumulation of garlic pathogenic bacteria, which is susceptible to complex infestation by multiple pathogenic bacteria. The applicant team can cause garlic root diseases and garlic red root diseases by collecting and evaluating garlic root rot pathogens in export garlic main producing areas in China, and besides common garlic root rot pathogens, the first time of finding a horn fungus (Ceratobasidium) strain DS54-3 (Y.S.Yin, J.J.Li, F.B.Zhang, S.Q.Zhang and M.Gao.first Report of Ceratobasidium sp.cause Root Rot of Garlic in China.plant diseases, 2020 104 (2): 569) hereinafter abbreviated as horn fungus, and a garlic root fungus (Setophoma terrestris) strain H5 (Zhang, F.B., zheng, H.L., cui, W.G., zhang, M.Q., yin, Y.S., cui, M., gao, M.first Report of Setophoma terrestris Causing Pink Root of Garlic in Chinese.plant diseases.2019, 103 (3), 584-584) which are the first time of finding garlic root diseases, have no effect of preventing and controlling garlic root fungi in garlic root diseases of 2 pathogenic fungi.

In recent years, the prevention and treatment of soil-borne diseases by using biological control technology has become a hot point of research at home and abroad, and many of the biological control fungi are trichoderma fungi, arbuscular mycorrhizal fungi, nonpathogenic fusarium oxysporum and the like. The biological control method utilizes some beneficial microorganisms to parasitize specific pathogenic bacteria in soil or generate harmful substances or reduce the number of pathogenic bacteria and infection of root systems by competing nutrition, space and other ways, and the antagonism method of bacterial control can avoid the harm brought by traditional agricultural control, chemical control and other methods, thus being a good control method for reducing the occurrence of diseases.

Disclosure of Invention

The invention aims to solve the technical problem of inhibiting pathogenic bacteria of garlic root rot and promoting garlic growth.

In order to solve the technical problems, the invention provides a strain Talaromyces liani which has inhibition effects on various pathogenic bacteria of garlic root rot, such as Fusarium oxysporum (Fusarium oxysporum), fusarium fingii (Fusarium redolens), basidiomycete (Ceratobasidium sp.), fusarium putrescens (Fusarium solani) and echinococcosis terrestris (Setophoma terrestris); meanwhile, the strain also has the function of promoting growth.

The strain number of Talaromyces liani provided by the invention is DS59-19F, and the registration number of the strain in the China general microbiological culture Collection center (CGMCC) is 23222.

Talaromyces liani DS59-19F has the ITS-rDNA sequence shown in SEQ ID No.7, the RPB2 gene sequence shown in SEQ ID No.8 and the CaM gene sequence shown in SEQ ID No. 9.

Cultures of Talaromyces liani DS59-19F are also within the scope of the invention.

The culture of Talaromyces liani DS59-19F provided by the invention is a substance obtained by culturing Talaromyces liani DS59-19F in a fungus culture medium (all substances in a culture container).

In the above cultures, the substances include metabolites of Talaromyces liani DS59-19F (the cells themselves) and Talaromyces liani DS59-19F.

The term "metabolite" refers to a primary metabolite and/or a secondary metabolite produced during metabolism of a microorganism. Primary metabolism refers to a process in which microorganisms absorb various nutrients from the outside and produce substances and energy that maintain vital activities through catabolism and anabolism. The primary metabolite is primary metabolite such as monosaccharide or monosaccharide derivative, nucleotide, vitamin, amino acid, fatty acid, and various macromolecular polymers composed of the same, such as protein, nucleic acid, polysaccharide, and lipid. Secondary metabolism refers to the process of synthesizing substances which have no definite function on the life activities of microorganisms by taking primary metabolites as precursors in a certain growth period of microorganisms. The secondary metabolite is the secondary metabolite, and most of the secondary metabolites are compounds with relatively complex molecular structures. Depending on their action, they can be classified into antibiotics, hormones, alkaloids, toxins, etc.

In the above culture, the fungus medium may be a solid medium or a liquid medium.

The term "culture" refers to a collective term for a liquid or solid medium in which a population of microorganisms has grown after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture. The term "culture" also includes subcultures obtained by passaging microorganisms, which may be a culture of a certain generation or a mixture of several generations.

In a specific embodiment of the invention, the fungal medium is in particular a PD medium or a PDA medium.

In order to solve the technical problems, the invention provides a microbial inoculum.

The microbial inoculum provided by the invention is a culture containing metabolites of Talaromyces liani DS59-19F and Talaromyces liani DS59-19F and/or Talaromyces liani DS59-19F.

Among the above-mentioned microbial agents, the microbial agents may be pathogen inhibitors, disease inhibitors, microbial agents for promoting plant growth and/or microbial agents for improving plant yield.

Among the microbial inoculum, the pathogenic bacteria inhibitor can have an inhibiting effect on pathogenic bacteria of garlic root rot; the disease may be garlic root rot.

Among the above bacterial agents, the pathogenic bacteria inhibitor may have an inhibitory effect on all or part of the following pathogenic bacteria of garlic root rot: fusarium oxysporum (Fusarium oxysporum), fusarium fingium (Fusarium redolens), talaromyces (Ceratobasidium sp.), fusarium putrescence (Fusarium solani) and Acidocella terrestris (Setophoma terrestris).

In a specific embodiment of the invention, the fusarium oxysporum (Fusarium oxysporum) is specifically fusarium oxysporum (Fusarium oxysporum) DS51-1F; the Fusarium fingium (Fusarium redolens) is specifically Fusarium fingium (Fusarium redolens) DS51-8F; the horn bacterium (Ceratobasidium sp.) is specifically a horn bacterium (Ceratobasidium sp.) DS54-3; the Fusarium solani (Fusarium solani) is specifically Fusarium solani (Fusarium solani) H9; the acanthocellum terrestris (Setophoma terrestris) is specifically acanthocellum terrestris (Setophoma terrestris) H5.

In the microbial inoculum, the active ingredients of the microbial inoculum can be metabolites of Talaromyces liani DS-19F and Talaromyces liani DS-19F and/or cultures of Talaromyces liani DS-19F, and the active ingredients of the microbial inoculum can also contain other biological ingredients or/and non-biological ingredients. Other active ingredients of the microbial inoculum can be determined by those skilled in the art according to antibacterial effect, disease resistance effect and plant growth promotion effect.

In the microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the pesticide arts and which is biologically inert. 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 above microbial inoculum, talaromyces liani DS-19F can be in the form of spores, mycelia or a culture containing spores and/or mycelia.

Among the above-mentioned bacterial agents, the formulation of the bacterial agent 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 pathogen inhibitors provided by the invention contain metabolites of Talaromyces liani DS59-19F, talaromyces liani DS59-19F, and/or cultures of Talaromyces liani DS59-19F.

Among the above agents, the metabolites of Talaromyces liani DS59-19F can be obtained from Talaromyces liani DS 59.sup.59-19F fermentation broth (e.g., sterile fermentation filtrate).

Any of the following uses for the metabolites of Talaromyces liani DS F-19F, talaromyces liani DS F59-19F and/or the cultures of Talaromyces liani DS F59-19F are also within the scope of the invention:

1) Inhibit pathogenic bacteria.

2) Preparing a pathogen inhibitor.

3) Inhibiting diseases.

4) And preparing a disease inhibitor.

Any of the following uses for the metabolites of Talaromyces liani DS F-19F, talaromyces liani DS F59-19F and/or the cultures of Talaromyces liani DS F59-19F are also within the scope of the invention:

a1 A product for promoting plant growth is prepared.

A2 Promoting plant growth.

A3 A product is produced that increases plant yield.

A4 Increasing plant yield.

Wherein the product may be an agricultural fertilizer.

In the above, the promotion of plant growth may be promotion of plant height increase, promotion of plant stem thickening increase, and/or promotion of plant fresh weight increase.

In the above, the plant may be any of the following:

p1) garlic;

p2) allium plants;

p3) plants of the family liliaceae.

The invention is Talaromyces liani DS-19F separated and screened from garlic rhizosphere soil in the export garlic main production area in China. Experiments prove that: the strain can antagonize 3 common garlic root rot pathogens and has obvious inhibition effect; meanwhile, the compound has obvious antagonism effect on pathogenic bacteria of garlic root rot, namely horn basidiomycete (Ceratobasidium sp.) and echinococcosis terrestris (Setophoma terrestris) which are discovered for the first time in China. The bacteriostatic rate of the Fusarium oxysporum (Fusarium oxysporum) DS51-1F reaches 72.81%, the bacteriostatic rate of the Fusarium oxysporum (Fusarium redolens) DS51-8F reaches 71.69%, the bacteriostatic rate of the Fusarium solani H9 reaches 78.81%, the bacteriostatic rate of the Fusarium oxysporum (Ceratobasidium sp.) DS54-3 reaches 66.67%, and the bacteriostatic rate of the Fusarium terrestris (Setophoma terrestris) H5 reaches 75.00%. As the garlic root rot is the compound infection of various pathogenic bacteria at present, the strain has wide application prospect in the aspect of preventing and controlling the garlic root rot as a biological pesticide.

Through a greenhouse cultivation test, the result shows that the garlic seedlings inoculated with the active bacterial agent-Talaromyces liani DS59-19F mycelium suspension are obviously different from the garlic seedlings inoculated with the same amount of clear water, and the Talaromyces liani DS-19F can obviously increase the indexes such as stem height, stem thickness, fresh weight and the like of garlic plants, and has the potential development capability of promoting the growth of the garlic seedlings and further improving the yield of garlic.

Preservation description

Classification naming: talaromyces liani;

biological materials according to: DS59-19F;

preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);

the preservation organization is abbreviated as: CGMCC;

address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;

preservation date: 2021, 8, 30;

accession numbers of the preservation center: CGMCC No.23222.

Drawings

FIG. 1 is a colony morphology observation of strain DS59-19F. A is the colony morphology after 3d incubation on OA, MEA, CYA and YES medium, respectively. B is an optical microscope observation chart.

FIG. 2 is a phylogenetic tree of the construction of isolates DS59-19F based on ITS, RPB2 and CaM genes.

FIG. 3 is a graph showing the effect of Talaromyces liani DS, 59-19F on the counter-action of various garlic pathogenic fungi. The pathogenic bacteria in A are Fusarium oxysporum (Fusarium oxysporum) DS51-1F; the pathogenic bacteria in the B are Fusarium fingium (Fusarium redolens) DS51-8F; the pathogenic bacteria in the C are horn bacillus (Ceratobasidium sp.) DS54-3; the pathogenic bacteria in the D are Fusarium solani (Fusarium solani) H9; the pathogenic bacteria in E are echinococcus terrestris (Setophoma terrestris) H5.

FIG. 4 is a graph showing the control effect of antagonizing fungi Talaromyces liani DS-19F in greenhouse water culture. The left side is the pathogenic bacteria echinocandin (Setophoma terrestris) H5 control (i.e., treatment 2); the middle part is simultaneously connected with the pathogenic bacteria of garlic root rot, namely echinococcus terrestris (Setophoma terrestris) H5 and antagonistic bacteria Talaromyces liani DS59-19F (namely treatment 3); on the right is a clear water control (i.e., treatment 1).

FIG. 5 is a greenhouse cultivation growth-promoting verification of Talaromyces liani DS59-19F. A is a graph showing the effect of inoculation 15D (negative control on the left; positive control on the middle; talaromyces liani DS59-19F.B on the right) and a graph showing the effect of potting 40D (negative control on the left; talaromyces liani DS-19F on the middle; positive control on the right), and it can be seen that the negative control plants have fallen.

Detailed Description

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.

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 and the like used in the examples described below are commercially available unless otherwise specified.

The media used in the examples below were as follows:

PD medium: cutting 200g of potato, boiling, filtering to obtain liquid, adding 20g of glucose, adding distilled water to 1L, sterilizing at 115 ℃ for 25min, and naturally pH.

PDA medium: cutting 200g of potato, boiling, filtering to obtain liquid, glucose 20g, agar 18g, adding distilled water to 1L, sterilizing at 115 deg.C for 25min, and naturally pH.

Soil leaching liquor culture medium: the soil was sieved (50 mesh) to 200g, 18g of agar, 1mL of 1% Bengalia red, and distilled water was used to fix the volume to 1L, pH7.0 or so. 0.1mL of 1% streptomycin was added to every 100mL of medium at the time of use.

OA medium: 30g of oatmeal, 18g of agar powder, adding distilled water to a constant volume of 1L, sterilizing at 115 ℃ for 30min, and naturally adjusting pH.

MEA medium: malt extract 20g, peptone 10g, glucose 20g, agar 20g, distilled water to 1L, sterilizing at 115 ℃ for 30min, and naturally pH.

CYA medium: 3.0g of sodium nitrate; 0.5g of potassium chloride; 0.5g of magnesium sulfate; ferrous sulfate 0.01g; 5.0g of yeast extract; 30.0g of sucrose; adding distilled water to a certain volume to 1L, sterilizing at 115 deg.C for 30min, and naturally maintaining pH.

YES medium: yeast extract powder 20g, sucrose 150g, mgSO ₄ ·7H ₂ 0.5g of O, 20g of agar, adding distilled water to a constant volume of 1L, sterilizing at 115 ℃ for 30min, and naturally adjusting pH.

Examples 1, talaromyces liani DS, 59-19F isolation and purification and identification

1. Isolation and purification of Strain DS59-19F

10g of soil sample (obtained from a plurality of garlic planting places 35.19 DEG N,116.38 DEG E, 35.21 DEG N,116.49 DEG E, 35.19 DEG N,116.23 DEG E, 35.03 DEG N,116.18 DEG E, jinxiang county, shandong Jining, main garlic producing area of China) was added to 100mL of sterile water, and the mixture was shake-cultured for 30 minutes to prepare a turbid liquid. 1mL of the above turbid solution was aspirated and added to a test tube containing 9mL of sterile water and mixed well (the dilution at this time was recorded as 10) ^-1 ) Then 1mL of the mixture is sucked from the test tube and added into another test tube containing 9mL of sterile water to be mixed uniformly, and the like is similarly prepared into 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 Bacterial suspensions of different dilutions. 0.1mL of each dilution is evenly coated on a PDA solid culture medium and a soil leaching solution culture medium plate, 3 parallels are made for each concentration, and the culture is carried out for 2-3 days at the constant temperature of 28 ℃. After colony formation, the colony is cultured in PDA culture medium plate or soil leaching liquid culture mediumAnd (3) carrying out multi-round strain purification on the plate to ensure that the strain is completely purified, wherein the total culture period is more than three weeks.

A total of 42 strains of fungi are separated and purified from a healthy garlic soil sample, wherein 18 strains are separated from a soil leaching solution culture medium, and 24 strains are separated from a PDA culture medium. The soil fungi are temporarily stored in a refrigerator with a PDA inclined plane at 4 ℃, and 5 parts of the soil fungi are respectively stored in an ultralow temperature refrigerator in 30% glycerol pipes.

The isolated 42 strains were initially screened for their antagonistic ability against a variety of garlic pathogenic fungi by a five-point counter method, and the results showed that the strain numbered DS59-19F had a good antagonistic effect against a variety of garlic root rot pathogenic fungi (see example 2 for details), and therefore the strain numbered DS59-19F was used for the following identification.

2. Identification of Strain DS59-19F

1. Morphological identification

Bacterial cakes with the same diameter and size are taken at the edge of a bacterial colony of which the bacterial strain DS59-19F is purified, and are placed in the center of a culture medium (OA, MEA, CYA and YES culture medium), and the growth rate of the bacterial strain, the growth condition, the bacterial colony color, the hypha morphological characteristics and other characters in the culture process are observed.

The result shows that after DS59-19F obtained by the separation and purification in the step one is cultured for 7d at 28 ℃, the colony is round, the surface is yellow, the edge is regular, the back is orange red, the surface is rough and has bulges, and hyphae extend to the periphery. No spores were found by microscopic examination (see FIG. 1).

2. Molecular biological identification

DNA extraction: scraping mycelia of a proper amount of strain DS59-19F, placing the mycelia into a sterile centrifuge tube, standing at the temperature of-80 ℃ for more than 30min, placing 1 steel ball into each centrifuge tube before starting DNA extraction, oscillating for 3min at the frequency of 50/60HZ by a freeze thawing cell disruptor, taking out the mycelia, sequentially performing the steps according to a fungus genome extraction kit (Beijing qing biological technology Co., ltd.) operation, collecting the DNA in a new centrifuge tube, measuring the concentration by using an ultra-micro spectrophotometer, and achieving the qualified DNA, and performing PCR amplification.

And (3) PCR amplification: ITS-rDNA segments are relatively conserved across a portion of fungi and are not sufficient to support the identification of species from some strains, and therefore some species require multiple gene integration. In this example, in addition to the identification of ITS, two genes, caM and RPB2, were identified, the primer sequences were as follows:

ITS-F：5’-TCCGTAGGTGAACCTGCGG-3’(SEQ ID No.1)；

ITS-R：5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID No.2)；

CaM-F：5’-GCCGACTCTTTGACYGARGAR-3’(SEQ ID No.3)；

CaM-R：5’-TTTYTGCATCATRAGYTGGAC-3’(SEQ ID No.4)；

RPB2-F：5’-GAYGAYMGWGATCAYTTYGG-3’(SEQ ID No.5)；

RPB2-R：5’-CCCATRGCTTGYTTRCCCAT-3’(SEQ ID No.6)。

in the above primer, Y is T or C; r is G or A; m is A or C; w is A or T.

PCR reaction system: 1. Mu.L of each of the upstream and downstream primers, 12.5. Mu.L of Taq PCR Starmix (with Loading Dye), 3. Mu.L of strain DNA template, ddH ₂ O7.5. Mu.L, 25. Mu.L overall.

ITS gene PCR amplification procedure: 94℃for 4min,94℃for 30s,53℃for 40s,72℃for 1min (30 cycles) and 72℃for 10min.

RPB2 gene PCR amplification procedure: 94℃2min,94℃1min,50℃2min,72℃3min (35 cycles), 72℃7min.

CaM gene PCR amplification procedure: 94℃1min,62℃30s,72℃90s (42 cycles), 72℃10min.

PCR product detection and sequencing: preparing agarose gel with the concentration of 1%, adding 1 mu L of DNA marker into the first or last gel hole, adding 2 mu L of PCR product into other gel holes, electrophoresis for 20min under 120V voltage, observing the condition of the strip under a gel imaging system, and collecting images.

After successful sample amplification, the samples were sent to a company (Shanghai Biotechnology Co., ltd.) for sequencing, the sequence of ITS-rDNA of strain DS59-19F was shown as SEQ ID No.7, the RPB2 gene was shown as SEQ ID No.8, and the CaM gene was shown as SEQ ID No. 9. The ITS-rDNA, RPB2 gene and CaM gene sequences of strain DS59-19F were aligned at NCBI Blast (https:// Blast. NCBI. Nrm. Nih. Gov/Blast. Cg), the relevant sequences of the model strain were downloaded, sequence evolution analysis was performed using MEGA7.0 software, and phylogenetic trees were constructed using the adjacent method (Neighbourj) to determine the classification status of the strain.

The results showed that the strains DS59-19F and Talaromyces liani CBS 225.66 were collected on the same branch, the relationship reached 98%, and the two strains and Talaromyces brevis CBS 141833 were collected on one large branch (see FIG. 2).

Through the morphological and molecular biological identification, the strain DS59-19F is identified as Talaromyces liani, and the strain is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC for short, with the address of Beijing Kogyo area North Chen Xiyi No. 1 and No. 3) in 2021, 8 months and 30 days, and the preservation number is CGMCC No.23222.

Examples 2, talaromyces liani DS59-19F plate counter verification experiment

The antagonistic ability of 42 fungi isolated in the step one of example 1 to a plurality of garlic pathogenic fungi was primarily screened by a five-point counter method, comprising: fusarium oxysporum (Fusarium oxysporum) DS51-1F, fusarium aromaticum (Fusarium redolens) DS51-8F, acidovorax (Ceratobasidium sp.) DS54-3, fusarium solani (Fusarium solani) H9, and Acidovorax faciens (Setophoma terrestris) H5. Among them, the pathogenic bacteria of garlic root rot (Ceratobasidium sp.) DS54-3 (described in "Y.S.Yin, J.J.Li, F.B.Zhang, S.Q.Zhang and M.Gao.first Report of Ceratobasidium sp.cause Root Rot of Garlic in China.plant Disease,2020 104 (2)", which is publicly available from the applicant, can only be used for repeating the experiment of the present invention, and cannot be used for him) and the pathogenic bacteria of garlic root rot (Acidovorax aculeatus (Setophoma terrestris) H5 (described in "Zhang, F.B., zheng, H.L., cui, W.G., zhang, M.Q., yin, Y.S., cui, M., & Gao, M.first Report of Setophoma terrestris Causing Pink Root of Garlic in China.plant Disease, 2019, 103 (3), which is publicly available from the applicant, can only be used for repeating the experiment of the present invention), are the pathogenic fungi of garlic root rot (not used for him) which are first reported by the team of garlic in China, and the pathogenic fungi of garlic root rot (2) are not discovered. Fusarium solani H9 is described in Wang Tian, etc. separation and pathogenicity preliminary study of pathogenic fungi of garlic root rot in garlic main producing areas in China, chinese soil and fertilizer 2020-12-08", available to the public from applicant, can be used only for repeating the experiment of the invention, and cannot be used for other purposes. The results show that: talaromyces liani DS59-19F has good antagonism effect on various garlic root rot pathogens.

The antagonism of Talaromyces liani DS-19F against a variety of garlic pathogenic fungi, including Fusarium oxysporum (Fusarium oxysporum) DS51-1F, fusarium fingii (Fusarium redolens) DS51-8F, fusarium carotovorum (Ceratobasidium sp.) DS54-3, fusarium solani (Fusarium solani) H9 and Cryptosporidium terrestris (Setophoma terrestris) H5 was further studied using a two-point counter method, and the antibacterial rate was calculated. Selecting a PDA plate, marking two dots at a position 2cm away from the center of the plate, inoculating one bacterial cake of Talaromyces liani DS-19F to one of the two dots, and inoculating one bacterial cake of garlic pathogenic fungi to the other dot. A control group (PDA plate is selected, two round dot marks are made at the position 2cm away from the center of the plate, one of which is inoculated with one bacterial cake of garlic pathogenic fungi, and the other is not inoculated with antagonistic bacteria) is arranged for each garlic pathogenic fungi. 3 replicates were set for each garlic pathogen fungus. After 7 days of culture at 28 ℃ in an incubator, the experimental result is observed, the experimental data is recorded by photographing, the antibacterial radius is measured, and the antibacterial rate is calculated.

Antibacterial ratio (%) = (control pathogen colony diameter-counter pathogen colony diameter)/control pathogen colony diameter x 100%.

The results show that the antibacterial rate of Talaromyces liani DS-19F against Fusarium oxysporum (Fusarium oxysporum) DS51-1F reaches 72.81%, the antibacterial rate against Fusarium oxysporum (Fusarium redolens) DS51-8F reaches 71.69%, the antibacterial rate against Fusarium (Certobiadium sp.) DS54-3 reaches 66.67%, the antibacterial rate against Fusarium solani (Fusarium solani) H9 reaches 78.81%, the antibacterial rate against Fusarium terrestris (Setophoma terrestris) H5 reaches 75.00%, and the specific plate counter situation is shown in FIG. 3.

Examples 3, talaromyces liani DS59-19F greenhouse hydroponic proofing verification

The pathogenic bacterial strain of the garlic red root rot selects the first reported pathogenic fungus of the garlic red root rot, namely echinococcosis terrestris (Setophoma terrestris) H5. Purified bacterial cakes of pathogenic bacteria of red root rot of garlic, namely echinococcosis terrestris (Setophoma terrestris) H5 and antagonistic bacteria Talaromyces liani DS-19F are respectively put into PD liquid culture medium, put into a shaking table and shake at the speed of 180r/min, and are cultivated for 7 days at the constant temperature of 28 ℃.

In a beaker filled with 600mL of tap water, healthy garlic which is free of damage, good in growth condition and consistent in size is picked up, sterilized by the surface of alcohol and placed on a buoy, so that the root is not in the tap water. Water is added on time to keep the water quantity stable. When garlic grows to seedling stage, experimental treatment is carried out, and spore liquid concentration (1×10) with optimal pathogenic bacteria is selected ⁸ cfu/mL) the concentration of antagonistic spores is regulated to be consistent with that of pathogenic bacteria (1 multiplied by 10) ⁸ cfu/mL), inoculating pathogenic bacteria by root injury pouring method after the antagonistic bacteria is colonized for 7d, and pouring the bacterial liquid into the roots of each plant after the roots are scratched by a sterile blade during inoculation, so as to pour clear water as a control. Treatment 1: comparing with clear water; treatment 2: inoculating control of Pythium gracile (Setophoma terrestris) H5; treatment 3: meanwhile, the pathogenic bacteria of garlic root rot, namely echinocandin (Setophoma terrestris) H5 and antagonistic bacteria Talaromyces liani DS-19F are inoculated. Each treatment set was repeated 5 times for 15 beakers, and garlic sensation and growth were observed periodically. The classification standard of root diseases in garlic sprout stage is as follows:

disease classification level 0:

aboveground part appearance: leaves are fresh green and have no pathological characteristics. Root expression: the garlic disk has luxuriant hair roots and no pathological characteristics.

Disease classification 1:

aboveground part appearance: only 1 leaf showed slight chlorosis, no more than 25% wilting and slightly softer stem base. Root expression: generally, the growth is good, the coverage area of the water-immersed disease spots is less than 15%, and the root number of the garlic is not obviously reduced.

Disease classification 3 grades:

aboveground part appearance: 1-2 leaves are yellowing, not more than 25% of plants with the leaf yellowing rate of more than 75% are wilting, and the basal part of the stem is water soaked. Root expression: the occurrence area of the water-immersed lesion is more than or equal to 15% and less than 50%, and the root is short and the hair root is reduced.

Disease classification 5 grades:

aboveground part appearance: 2-3 leaves turn yellow, plants are short and have wilting of more than 75%, and the base of the stem is soft and is in brown water. Root expression: the occurrence area of the water-immersed disease spots is more than 50%, the root is short, crisp and easy to break, the garlic disk is light brown, and the root growing amount is obviously reduced.

Disease classification 7:

aboveground part appearance: all leaves become withered and yellow, all plants wilt short and the stem base is seriously rotten. Root expression: the water stain disease spots cover the whole root system, and the garlic discs do not develop roots.

The pathogenesis calculation formula is as follows:

disease index = (Σmulti-disease number x number of disease plants)/(total number of investigation x highest number) x 100 (Zhang Bo, 2008, preliminary study of garlic pythium root rot in shandong province. Agricultural university in Xinjiang).

Control effect= (control incidence index-treatment incidence index)/control incidence index x 100% (Zhang Bo, 2008, preliminary study of garlic pythium root rot in shandong province. University of agriculture in Xinjiang).

The result shows that the single pathogen starts to cause symptoms of garlic diseases after inoculation for about 7d, and the symptoms of garlic red root rot which are consistent with the pathogenicity measurement phenomenon of echinococcus terrestris (Setophoma terrestris) H5 are shown, wherein the symptoms of garlic red root rot are shown, and the disease index is as high as 88.6%; the treatment of the access antagonistic fungi Talaromyces liani DS-19F has obvious prevention and control effects compared with a pathogenic bacteria control group, has growth vigor difference compared with a clear water control group, but most plant roots are healthy, the disease index is 36.2%, most of the plants show 3-level symptoms, the control is more than 5-level and above, the prevention and control effects reach 59.1%, and the greenhouse water culture effect is shown in figure 4.

Examples 4, talaromyces liani DS59-19F greenhouse cultivation growth-promoting verification

Talaromyces liani DS59-19F and positive control industrial fungus Trichoderma harzianum ACCC 30371 (from China center for type culture collection of microorganisms) are respectively inoculated into PD culture media, and subjected to shaking culture at 180r/min and at 28 ℃ for 5 days. Preparation of Talaromyces liani DS59-19F mycelium suspension with PD Medium at a concentration of 1X 10 ⁸ cfu/mL is reserved. The garlic is planted in a nutrition matrix after being disinfected, 3 garlic is placed in a greenhouse after each basin, and is watered every day without using fertilizer. And when the garlic seedlings grow to about 10cm, irrigating the prepared mycelium suspension liquid around the garlic, wherein each pot is 50mL, and irrigating clear water is used as negative control. After 40 days of planting, the phenomenon was observed and the index was measured.

In the initial stage of the experiment, the inoculated Talaromyces liani DS-19F and the negative control group have no obvious difference, after 15 days, the negative control group without the microbial inoculum gradually has the leaf-hanging phenomenon, the later stage has the leaf-yellowing phenomenon, the watering industrial fungus Trichoderma harzianum ACCC 30371 positive control group has no obvious leaf-hanging phenomenon, but the plant is relatively short and small, and the later stage has the leaf-hanging phenomenon. And is shown in fig. 5. Through practical measurement, the plant inoculated with Talaromyces liani DS59-19F is obviously higher than the negative control group (P < 0.05) in plant height, stem thickness and fresh weight indexes, which shows that the fungus Talaromyces liani DS59-19F has obvious growth promoting effect on garlic seedlings in a greenhouse cultivation stage, and the specific data are as follows:

negative control group:

fresh weight of plant (g): 4.22.+ -. 0.51. Plant height (cm): 45.33+ -3.03. Stem thickness (cm): 0.55 + -0.05.

Positive control group:

fresh weight of plant (g): 8.01 + -0.83. Plant height (cm): 54.54.+ -. 2.36. Stem thickness (cm): 0.65 + -0.04.

Talaromyces liani DS59 groups 59-19F:

fresh weight of plant (g): 8.07 + -0.96. Plant height (cm): 57.25.+ -. 2.29. Stem thickness (cm): 0.66 + -0.55.

The values of plant height, stem thickness and plant fresh weight in each of the above groups are expressed as "average ± standard deviation".

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. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> institute of agricultural resource and agricultural division of national academy of agricultural sciences

<120> a fungus having antagonistic action against pathogenic bacteria of garlic root rot and growth promoting effect

<130> GNCLN212792

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 19

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<213> Artificial sequence

<400> 1

tccgtaggtg aacctgcgg 19

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

tcctccgctt attgatatgc 20

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<211> 21

<212> DNA

<213> Artificial sequence

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gccgactctt tgacygarga r 21

<210> 4

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tttytgcatc atragytgga c 21

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gaygaymgwg atcayttygg 20

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cccatrgctt gyttrcccat 20

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<212> DNA

<213> Talaromyces liani

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tttggcgggc ccaccggggc cacctggtcg ccgggggacg cacgtccccg ggcccgcgcc 120

cgccgaagcg cgctgtgaac cctgatgaag atgggctgtc tgagtactat gaaaattgtc 180

aaaactttca acaatggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga 240

taagtaatgt gaattgcaga attccgtgaa tcatcgaatc tttgaacgca cattgcgccc 300

cctggcattc cggggggcat gcctgtccga gcgtcatttc tgccctcaag cacggcttgt 360

gtgttgggtg tggtcccccc ggggacctgc ccgaaaggca gcggcgacgt ccgtctggtc 420

ctcgagcgta tggggctctg tcactcgctc gggaaggacc tgcgggggtt ggtcaccacc 480

acattttacc acggttgacc tcggatcagg taggagttac ccgctgaact taagcatatc 540

aataagcgga ggaa 554

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atctcacccg ttacgtccag agatgcgttg aaacaaaccg tgaagtggtt ctcaacgtgg 60

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gtgagcagaa gaaggcaatg agctcgaaag caggtgtttc tcaggtgctc agtcgataca 180

cctttgcctc tactttgtct catttaagac gtaccaatac gcctattggc cgtgatggga 240

aaatcgccaa gcctcgtcag ctacataaca ctcactgggg tctggtttgt cctgccgaga 300

ctcctgaagg tcaagcttgt ggtttggtca aaaacttggc tttgatgtgt tctatcactg 360

tgggttctcc tagcgagcct attgttgatt tcatgattca acgaaacatg gaagtgcttg 420

aagaattcga accgctagtt acacctcatg ccactaaggt ctttgtcaat ggtgtttggg 480

ttggtgtgca tcgtgacccg gctcatttgg tcagcactgt ccagtcacta cgccgacgga 540

atatgatttc ccacgaagtc agcttagttc gtgatattcg tgaccgagag ttcaagatct 600

tcacagatgc tggtcgtgtt tgtcgaccac ttttcgtcat tgacaacgat ccacgaagtg 660

aaaactgcgg atctttggtg ctcaacaaag accatattcg cagacttgaa gcagaccgcg 720

agcttccacc agacctcgac cccgaagaac gaagggaaca gtactatggc tgggagggtc 780

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agggttatcg ccgcgaacag atattgacta tatcgaatag gtcaaatcac aaccaaggaa 180

ctgggcactg tcatgcgttc cctcggccag aacccctccg aatccgaact gcaggacatg 240

atcaacgaag tcgacgctga caacaacggc acaatcgatt tccctggtat gatataattg 300

ctcacaggat attatgatgg cagtactaac tgccgcagaa ttcctgacaa tgatggcccg 360

caaaatgaag gacaccgact ccgaggaaga gatccgcgag gcattcaagg tgtttgaccg 420

tgacaacaat ggattcatct ctgctgccga attgcgccac gttatgacct cgattggcga 480

gaagttgacc gacgacgagg ttgatgagat gattcgcgag gctgaccagg acggtgatgg 540

aaggattgac tgtaagcctc gctcgctgtc tcttcatgaa atcagaacac cattctaaca 600

tgcgataatt aga 613

Claims (8)

1.Talaromyces lianiThe strain number is DS59-19F, and the registration number of the strain in the China general microbiological culture Collection center is CGMCC No.23222.

2. The microbial inoculum is characterized in that: the microbial inoculum comprising the composition according to claim 1Talaromyces liani。

3. The microbial agent of claim 2, wherein: the microbial inoculum is a pathogenic bacteria inhibitor, a disease inhibitor, a microbial inoculum for promoting plant growth and/or a microbial inoculum for improving plant yield.

4. A microbial agent according to claim 3, wherein: the pathogenic bacteria inhibitor has an inhibiting effect on pathogenic bacteria of garlic root rot; the disease is garlic root rot.

5. The microbial agent of claim 4, wherein: the pathogen inhibitor has an inhibiting effect on all or part of the following garlic root rot pathogens: fusarium oxysporum, fusarium fingium, fusarium carotovorum, fusarium putrescens and Acidocella terrestris.

6. A microbial agent according to claim 3, wherein: the plant is any one of the following:

p1) garlic;

p2) allium plants;

p3) plants of the family liliaceae.

7. The method of claim 1Talaromyces lianiAny one of the following applications:

1) Inhibiting pathogenic bacteria;

2) Preparing a pathogenic bacteria inhibitor;

3) Inhibiting diseases;

4) Preparing a disease inhibitor;

the pathogenic bacteria are pathogenic bacteria of garlic root rot; the disease is garlic root rot.

8. The method of claim 1Talaromyces lianAnd/or any one of the following applications of the microbial agent of any one of claims 2-5:

a1 Preparing a product for promoting plant growth;

a2 Promoting plant growth;

a3 Preparing a product that increases plant yield;

a4 Increasing plant yield;

the plant is garlic.

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