CN114032182A

CN114032182A - Fungus with functions of antagonizing garlic root rot pathogenic bacteria and promoting growth

Info

Publication number: CN114032182A
Application number: CN202111412503.6A
Authority: CN
Inventors: 高淼; 任东军; 尹彦舒; 魏海雷
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-02-11
Anticipated expiration: 2041-11-25
Also published as: CN114032182B

Abstract

The invention discloses a fungus strain with functions of antagonizing garlic root rot pathogenic bacteria 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 is CGMCC No. 23222. The strain can simultaneously antagonize various garlic root rot pathogenic bacteria, including the first garlic root rot pathogenic bacteria, namely rhizopus angulatus (Ceratobasidium sp) and acanthosporium terrestris (Setophorum terrestris) found in China. 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 seedling growth so as to improve garlic yield.

Description

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

Technical Field

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

Background

Garlic is a perennial herb of the genus Allium in the family Liliaceae, is one of the spicy vegetables which people in China love, and is also an important economic crop in China. With the increasing demand of garlic, the planting area and the continuous planting life of garlic are continuously increased, so that continuous cropping obstacles, namely Soil decline (Soil shock), are shown as outbreaks of garlic root rot, and a plurality of serious problems are brought to agricultural production, ecological environment and food safety. Research finds that the garlic root rot pathogenic bacteria in China mainly comprise a plurality of pathogenic bacteria such as Fusarium oxysporum (Fusarium oxysporum), Fusarium proliferatum (Fusarium proliferatum), Fusarium reoccum (Fusarium redolen), Pythium (Phthium) and the like; research also finds that the garlic or other allium crops planted continuously can cause the accumulation of garlic pathogenic bacteria and are easy to cause compound infection of various pathogenic bacteria. By collecting and evaluating the Garlic Root Rot pathogenic bacteria in the major Garlic producing area exported by our country, the applicant found that the fungi of the genus Ceratobasidium (Ceratobasidium) strain DS54-3(y.s.yin, j.j.li, f.b.zhang, s.q.zhang and m.gao.first Report of Ceratobasidium sp.using Root of Garlic in china plant Disease, 2020104 (2):569) are hereinafter referred to as angle-carrierella, and the fungus of the species echinospora terrestris H5(Zhang, f.b., Zheng, h.l., Cui, w.g., Zhang, m.q., Yin, y.s.s., Cui, m.g., Zhang, m.q., Yin, g., garesium, g., garesium 584, field of Garlic spot 584, which has no effect on controlling Garlic Root Rot pathogenic bacteria in the fungi of the country, and the fungus of the Garlic Root Rot pathogenic bacteria of the country are not reported (Garlic Root Rot pathogen) in the group).

In recent years, the prevention and control of soil-borne diseases by using a biological prevention and control technology becomes a hot spot of domestic and foreign research, and trichoderma fungi, arbuscular mycorrhizal fungi, nonpathogenic fusarium oxysporum and the like are researched and applied in biological prevention and control fungi. The biological control is to reduce the number of pathogenic bacteria and the infection of root systems by utilizing the parasitism of some beneficial microorganisms to specific pathogenic bacteria in soil or generating harmful substances or competing the ways of nutrition, space and the like, the antagonism method for controlling bacteria by bacteria can avoid the harm brought by the traditional methods of agricultural control, chemical control and the like, and is a good control method for reducing the occurrence of diseases.

Disclosure of Invention

The invention aims to solve the technical problem of how to inhibit the pathogenic bacteria of garlic root rot and promote the growth of garlic.

In order to solve the technical problems, the invention provides a Talaromyces liani, which has an inhibiting effect on various garlic root rot pathogenic bacteria, such as Fusarium oxysporum (Fusarium oxysporum), Fusarium fragrans (Fusarium redolen), Rhizoctonia solani (Ceratobasidium sp.), Fusarium solani (Fusarium solani) and Echizomyces terrestris (Setophorus terrestris); meanwhile, the strain also has a growth promoting function.

The strain number of the Talaromyces liani provided by the invention is DS59-19F, and the registration number of the Talaromyces liani in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No. 23222.

Talaromyces liani DS59-19F has an ITS-rDNA sequence shown in SEQ ID No.7, an RPB2 gene sequence shown in SEQ ID No.8 and a 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 (all substances in a culture container) obtained by culturing Talaromyces liani DS59-19F in a fungus culture medium.

In the above culture, the substance includes Talaromyces liani DS59-19F (bacterial cells themselves) and a metabolite of Talaromyces liani DS 59-19F.

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

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

The term "culture" refers to a general term for liquid or solid medium on which a microorganism population grows after artificial inoculation and culture. I.e. a product obtained by growing and/or amplifying a microorganism, which may be a biologically pure culture of the microorganism, or which may contain a certain amount of a culture medium, metabolite or other component produced during the cultivation. The term "culture" also includes subcultures obtained by passaging the microorganism, which may be a generation of culture or a mixture of generations.

In a particular embodiment of the invention, the fungal culture 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 Talaromyces liani DS59-19F, a metabolite of Talaromyces liani DS59-19F and/or Talaromyces liani DS 59-19F.

In the microbial inoculum, the microbial inoculum can be a pathogenic bacteria inhibitor, a disease inhibitor, a microbial inoculum for promoting plant growth and/or a microbial inoculum for improving plant yield.

In the microbial inoculum, the pathogenic bacteria inhibitor can inhibit garlic root rot pathogenic bacteria; the disease may be garlic root rot.

In the microbial inoculum, the pathogenic bacteria inhibitor can inhibit all or part of the following garlic root rot pathogenic bacteria: fusarium oxysporum (Fusarium oxysporum), Fusarium fragrans (Fusarium redolen), Fusarium graminearum (Ceratobasidium sp.), Fusarium solani (Fusarium solani), and Echizomucopias terrestris (Setophoma terrestris).

In a particular embodiment of the invention, the Fusarium oxysporum (Fusarium oxysporum) is in particular Fusarium oxysporum (Fusarium oxysporum) DS 51-1F; the Fusarium fragrans (Fusarium redolen) is specifically Fusarium fragrans (Fusarium redolen) DS 51-8F; the basidiomycete (Ceratobasidium sp.) is specifically basidiomycete (Ceratobasidium sp.) DS 54-3; the Fusarium solani (Fusarium solani) is specifically Fusarium solani (Fusarium solani) H9; the Echinospora terrestris (Setophoma terrestris) is specifically Echinospora terrestris (Setophoma terrestris) H5.

In the microbial inoculum, the active ingredients of the microbial inoculum can be Talaromyces liani DS59-19F, metabolites of Talaromyces liani DS59-19F and/or cultures of Talaromyces liani DS59-19F, 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 the technicians in the field according to the bacteriostatic effect, the disease-resistant effect and the plant growth promoting effect.

In the above microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be one that is commonly used in the pesticide art and is biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound can be polyvinyl alcohol and/or polyglycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane.

Among the above microbial agents, Talaromyces liani DS59-19F may exist in the form of spores, hyphae, or a culture containing spores and/or hyphae.

In the microbial inoculum, the dosage form of the microbial inoculum can be various dosage forms, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The pathogen inhibitor provided by the invention contains Talaromyces liani DS59-19F, metabolites of Talaromyces liani DS59-19F, and/or cultures of Talaromyces liani DS 59-19F.

Among the above microbial agents, metabolites of Talaromyces liani DS59-19F can be obtained from fermentation broth of Talaromyces liani DS59-19F (such as sterile fermentation filtrate).

Also within the scope of the present invention is any of the following uses of Talaromyces liani DS59-19F, a metabolite of Talaromyces liani DS59-19F, and/or a culture of Talaromyces liani DS 59-19F:

1) inhibiting pathogenic bacteria.

2) Preparing a pathogenic bacteria inhibitor.

3) And (5) disease inhibition.

4) Preparing a disease inhibitor.

A1) preparing the product for promoting the growth of plants.

A2) Promoting the growth of plants.

A3) Produce products that increase plant yield.

A4) The yield of the plant is improved.

Wherein the product may be an agricultural fertilizer.

As described above, the promotion of plant growth may be the promotion of an increase in plant height, the promotion of an increase in stem thickness of a plant, and/or the promotion of an increase in fresh weight of a plant.

Above, the plant may be any one of:

p1) garlic;

p2) plants of the allium genus;

p3) plants of the liliaceae family.

The invention relates to Talaromyces liani DS59-19F obtained by separating and screening garlic rhizosphere soil in a main garlic producing area exported in China. Experiments prove that: the strain can antagonize 3 common garlic root rot pathogenic bacteria and has obvious inhibition effect; meanwhile, the composition has obvious antagonistic effect on pathogenic bacteria of garlic root rot (Ceratobasidium sp) and Echinospora terrestris (Setophora terrestris) which are firstly discovered in China. The bacteriostatic rate of the compound has the advantages that the bacteriostatic rate of the compound on Fusarium oxysporum DS51-1F reaches 72.81 percent, the bacteriostatic rate on aromatic Fusarium (Fusarium redolen) DS51-8F reaches 71.69 percent, the bacteriostatic rate on Fusarium solani H9 reaches 78.81 percent, the bacteriostatic rate on Rhizoctonia solani (Ceratobasidium sp) DS54-3 reaches 66.67 percent, and the bacteriostatic rate on Echinospora terrestris (Setophorus terrestris) H5 reaches 75.00 percent. Because 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 treating the garlic root rot as a biological pesticide.

Through greenhouse cultivation experiments, the garlic seedlings inoculated with an active microbial inoculum, namely Talaromyces liani DS59-19F hypha suspension, are found to have significant difference with garlic seedlings inoculated with the same amount of clear water, and the results show that the Talaromyces liani DS59-19F can significantly increase indexes such as stem height, stem thickness and fresh weight of garlic plants, and has the potential development capacity of promoting the growth of the garlic seedlings so as to improve the garlic yield.

Deposit description

And (3) classification and naming: talaromyces liani;

the biological material of the reference: DS 59-19F;

the preservation organization: china general microbiological culture Collection center;

the preservation organization is abbreviated as: CGMCC;

address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North;

the preservation date is as follows: 8, month and 30 days 2021;

registration number of the preservation center: CGMCC No. 23222.

Drawings

FIG. 1 is a colony morphology observation of strain DS 59-19F. A is colony morphology after 3d of culture on OA, MEA, CYA and YES medium, respectively. B is an optical microscope observation image.

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

FIG. 3 is a graph showing the effect of Talaromyces liani DS59-19F on various garlic pathogenic fungi. The pathogenic bacteria in A is Fusarium oxysporum (Fusarium oxysporum) DS 51-1F; the pathogenic bacteria in B is aromatic Fusarium (Fusarium redolens) DS 51-8F; the pathogenic bacteria in C is a basidiomycetes (Ceratobasidium sp) DS 54-3; the pathogenic bacteria in D is Fusarium solani (Fusarium solani) H9; the pathogenic bacteria in E is Echinospora terrestris (Setophoma terrestris) H5.

FIG. 4 is a diagram of the greenhouse water culture prevention and control effect of the antagonistic fungus Talaromyces liani DS 59-19F. Left hand side is control of pathogenic bacteria Echinospora terrestris (Setophoma terrestris) H5 (i.e., treatment 2); the middle is simultaneously inoculated with garlic root rot pathogenic bacteria Echinospora terrestris H5 and antagonistic bacteria Talaromyces liani DS59-19F (namely treatment 3); the right side is the clear water control (i.e., treatment 1).

FIG. 5 is a diagram showing the growth of Talaromyces liani DS59-19F in greenhouse cultivation. A is an effect graph of inoculation 15D (negative control on the left, positive control in the middle, and Talaromyces liani DS59-19 F.B on the right) for 40D (negative control on the left, Talaromyces liani DS59-19F in the middle, and positive control on the right), and the negative control plant is seen to have fallen, C is a comparison graph of the positive control bacterium Trichoderma harzianum ACCC 30371 with the negative control without the addition of the fungicide (inoculation 40D) (positive control on the left, negative control on the right), D is a comparison graph of Talaromyces liani DS59-19F with the negative control without the addition of the fungicide (inoculation 40D) (Talaromyces liani DS59-19F on the left, and negative control on the right).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The media used in the following examples are as follows:

PD medium: cutting potato 200g into pieces, boiling, filtering to obtain liquid, adding glucose 20g, adding distilled water to desired volume of 1L, sterilizing at 115 deg.C for 25min, and keeping pH natural.

PDA culture medium: cutting 200g of potato into pieces, boiling, filtering to obtain liquid, 20g of glucose and 18g of agar, adding distilled water to a constant volume of 1L, sterilizing at 115 ℃ for 25min, and keeping the pH value natural.

Soil extract culture medium: the soil was sieved (50 mesh) 200g, agar 18g, 1% Bengal red 1mL, and the volume was adjusted to 1L using distilled water, 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 and 18g of agar powder, adding distilled water to a constant volume of 1L, sterilizing at 115 ℃ for 30min, and keeping the pH natural.

MEA culture medium: 20g of malt extract powder, 10g of peptone, 20g of glucose and 20g of agar, adding distilled water to a constant volume of 1L, sterilizing at 115 ℃ for 30min, and naturally adjusting the pH value.

CYA medium: 3.0g of sodium nitrate; 0.5g of potassium chloride; magnesium sulfate 0.5 g; 0.01g of ferrous sulfate; 5.0g of yeast extract; 30.0g of cane sugar; adding distilled water to desired volume of 1L, sterilizing at 115 deg.C for 30min, and keeping pH natural.

YES medium: 20g of yeast extract powder, 150g of cane sugar and MgSO₄·7H₂0.5g of O and 20g of agar, and adding distilled water to a constant volume of 1L and 115 DEG CSterilizing for 30min, and keeping pH natural.

Example 1 isolation, purification and characterization of Talaromyces liani DS59-19F

Firstly, separating and purifying strain DS59-19F

10g of soil samples (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 and 116.18 DEG E from a plurality of garlic planting fields in Jinxiang county, Jining City, Shandong province, Ministry of major garlic production in China) were added to 100mL of sterile water and subjected to shaking culture for 30min to prepare a turbid solution. Sucking 1mL of the turbid solution, adding the turbid solution into a test tube containing 9mL of sterile water, and fully mixing the turbid solution uniformly (the dilution is recorded as 10 in the moment)^-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 uniformly mixed, and the like is repeated to prepare 10^-2、10^-3、10^-4、10^-5Bacterial suspensions of various dilutions. 0.1mL of each dilution is uniformly coated on a PDA solid culture medium and a soil extract culture medium plate, each concentration is performed in 3 parallel, and the constant temperature of 28 ℃ is kept for static culture for 2 to 3 days. After the colony is formed, performing multiple rounds of strain purification on a PDA culture medium plate or a soil extract culture medium plate to completely purify the strain, wherein the total culture period is more than three weeks.

And (3) separating and purifying the total 42 fungi from a healthy garlic soil sample, wherein 18 fungi are separated on a soil extract culture medium, and 24 fungi are separated on a PDA culture medium. Temporarily storing the soil fungi in a PDA inclined plane refrigerator at 4 ℃, and simultaneously storing 5 parts of the soil fungi in an ultralow temperature refrigerator in 30% glycerin pipes respectively.

The five-point antagonism method is adopted to preliminarily screen the antagonistic capability of the separated 42 strains on various garlic pathogenic fungi, and the result shows that the strains with the numbers of DS59-19F have better antagonistic effect on various garlic root rot pathogenic fungi (see example 2 for details), so the strains with the numbers of DS59-19F are taken for the following identification.

II, identifying strain DS59-19F

1. Morphological identification

And (3) taking fungus cakes with the same diameter at the edges of purified colonies of the strain DS59-19F, placing the fungus cakes in the center of a culture medium (OA, MEA, CYA and YES culture media), and observing the growth rate of the strain and the properties such as the growth condition, the colony color, the hypha morphological characteristics and the like in the culture process.

The result shows that after the DS59-19F separated and purified in the first step is cultured at 28 ℃ for 7 days, the colony is circular, the surface is yellow, the edge is regular, the back surface is orange red, the surface is rough and has bulges, and hyphae extend to the periphery. No spores were found by microscopy (see figure 1).

2. Molecular biological identification

DNA extraction: scraping a proper amount of hyphae of a strain DS59-19F, placing the hyphae in a sterile centrifuge tube at-80 ℃ for more than 30min, before DNA extraction, placing 1 steel ball in each centrifuge tube, oscillating the hyphae for 3min at the frequency of 50/60HZ by a freeze-thaw cell disruptor, taking out the hyphae, sequentially performing the steps according to the operation steps of a fungus genome extraction kit (Beijing engine Biotechnology Co., Ltd.), collecting DNA in a new centrifuge tube, measuring the concentration by using an ultramicro spectrophotometer, and performing PCR amplification on qualified DNA.

And (3) PCR amplification: the ITS-rDNA segment is relatively conservative for a part of fungi and is not enough to support the identification result of some strains to determine species, so that some species need multiple gene comprehensive identification. In this example, in addition to the identification of ITS, two genes, CaM and RPB2, were identified, with the following primer sequences:

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.

And (3) PCR reaction system: 1 μ L of each of the upstream and downstream primers, 12.5 μ L of Taq PCR StarMix (with Loading Dye), 3 μ L of strain DNA template, ddH₂O7.5. mu.L, Total 25μL。

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

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

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

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

After the sample is successfully amplified, the sample is sent to a company (Shanghai Biotechnology engineering Co., Ltd. carries out sequencing, the ITS-rDNA sequence of the strain DS59-19F is shown as SEQ ID No.7, the RPB2 gene is shown as SEQ ID No.8, and the CaM gene is shown as SEQ ID No. 9. the ITS-rDNA, the RPB2 gene and the CaM gene sequence of the strain DS59-19F are compared in NCBI Blast (https:// blast.ncbi.n.lm.nih.gov/blast.cg), the related sequences of the model strain are downloaded, the sequence evolution analysis is carried out by using MEGA7.0 software, and a phylogenetic tree is constructed by using a neighbor method (N-J) to determine the classification status of the strain.

The results show that the strain DS59-19F and Talaromyces liani CBS 225.66 are clustered on the same branch, the genetic relationship reaches 98%, and the two strains and Talaromyces brevis CBS 141833 are clustered on a large branch (see figure 2).

Through the above morphological and molecular biological identification, the strain DS59-19F is identified as Talaromyces liani, which has been deposited in China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, CGMCC for short, address: No. 3, Xilu 1, Beijing, the sunward, 30), at 8 months and 30 days in 2021, and the deposit number is CGMCC No. 23222.

Example 2 Talaromyces liani DS59-19F plate confrontation verification experiment

Adopting five-point confronting method, preliminarily screening the antagonistic capacity of 42 strains of the fungus separated in the first step of the example 1 on various garlic pathogenic fungi, comprising the following steps: fusarium oxysporum (Fusarium oxysporum) DS51-1F, Fusarium fragrans (Fusarium redolen) DS51-8F, Fusarium graminearum (Ceratobasidium sp.) DS54-3, Fusarium solani (Fusarium solani) H9, and Echium terrestris (Setophoma terrestris) H5. Among them, Garlic Root Rot pathogen angular strain (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.using Root Rot of Garlic in china plant Disease, 2020104 (2): 569" a document publicly available from the applicant and only available for use in the duplicate invention experiment, not others) and Garlic red Root Rot pathogen echinococcus terrestris (setophor) H5 (described in "Zhang, f.b., Zheng, h.l., Cui, w.g., Zhang, m.q. Yin, y.s., Cui, garsic, m.584, m.g., in the public defense against garrisonia pathogen, such as found in the public defense team in Garlic plant Disease, such as garrisonia bacterium 584, the first Report that the Garlic red Root Rot pathogen is available from the applicant and only available for use in the duplicate invention (first Report that the Garlic red Root Rot pathogen in china plant Disease pathogen free from garrisonia plant Disease team 103). Fusarium solani (Fusarium solani) H9 is described in Wantian sweet et al, separation of pathogenic fungi of garlic root rot in main garlic producing areas in China and preliminary study on pathogenicity, China soil and fertilizer, 2020-12-08, the public can obtain from the applicant, can only be used for repeated experiments, and cannot be used for other purposes. The results show that: talaromyces liani DS59-19F has better antagonistic effect on various garlic root rot pathogenic bacteria.

The antagonistic ability of Talaromyces liani DS59-19F against various garlic pathogenic fungi including Fusarium oxysporum DS51-1F, Fusarium reolenum DS51-8F, Fusarium angulatum (Ceratobasidium sp.) DS54-3, Fusarium solani (Fusarium solani) H9 and Echinosporium terrae (Setophorum terrestris) H5 was further studied by two-point confrontation, and the antibacterial rate was calculated. Selecting a PDA (personal digital assistant) plate, marking two round dots at the position 2cm away from the center of the plate, inoculating one fungus cake of Talaromyces liani DS59-19F on one point, and inoculating one fungus cake of garlic pathogenic fungi on the other point. A control group is set for each garlic pathogenic fungus (a PDA flat plate is selected, two round dot marks are made at the position 2cm away from the center of the flat plate, one dot is inoculated with a fungus cake of the garlic pathogenic fungus, and the other dot is not inoculated with antagonistic bacteria). 3 replicates were set for each garlic pathogenic fungus. After culturing for 7 days at 28 ℃ in an incubator, observing the experimental result, photographing and recording experimental data, measuring the bacteriostatic radius of the experimental data, and calculating the bacteriostatic rate.

The bacteriostatic ratio (%) - (control pathogen colony diameter-opposing pathogen colony diameter)/control pathogen colony diameter × 100%.

The results show that the inhibition rate of Talaromyces liani DS59-19F on Fusarium oxysporum (Fusarium oxysporum) DS51-1F reaches 72.81%, the inhibition rate on aromatic Fusarium (Fusarium redolen) DS51-8F reaches 71.69%, the inhibition rate on Rhizoctonia solani (Ceratobasidium sp.) DS54-3 reaches 66.67%, the inhibition rate on Fusarium solani (Fusarium solani) H9 reaches 78.81%, the inhibition rate on Rhizoctonia terrestris (Setophorus terrestris) H5 reaches 75.00%, and the specific plate facing condition is shown in FIG. 3.

Example 3 Talaromyces liani DS59-19F greenhouse water culture control effect verification

The pathogenic strain of garlic red root rot is selected from the first domestic reported pathogenic fungus of garlic red root rot, i.e. Echinospora terrestris (Setophoma terrestris) H5. Respectively pouring purified cakes of pathogenic bacteria of garlic red root rot, namely Echinospora terrestris (Setophoresis) H5 and antagonistic bacteria Talaromyces liani DS59-19F into a PD liquid culture medium, putting into a shaking table, shaking at the speed of 180r/min, and culturing at the constant temperature of 28 ℃ for 7 days.

In a beaker filled with 600mL of tap water, healthy garlic which is not damaged, has good growth condition and consistent size is picked, sterilized by the surface of alcohol and then placed on a buoy, so that the root is immersed in the tap water. Water is added on time to keep the water quantity stable. When the garlic grows to the seedling stage, the experimental treatment is carried out, and the concentration (1 multiplied by 10) of spore liquid with the best pathogenic bacteria is selected⁸cfu/mL), the concentration of the antagonistic bacteria spores is adjusted to be consistent with that of the pathogenic bacteria (1 multiplied by 10)⁸cfu/mL), inoculating pathogenic bacteria by adopting a damaged root perfusion method after the antagonistic bacteria is colonized for 7dWhen inoculating, the root of each plant is scratched by a sterile blade, and then the root of each plant is irrigated by bacterial liquid, and clear water is irrigated as a control. Treatment 1: clear water control; and (3) treatment 2: inoculating control of Echinospora terrestris (Setophoma terrestris) H5; and (3) treatment: simultaneously inoculating the garlic root rot pathogen Echinospora terrestris H5 and antagonistic bacteria Talaromyces liani DS 59-19F. Each group of treatment is provided with 5 repetitions, and 15 beakers are used for treatment, and garlic infection and growth conditions are observed regularly. The grading standard of the garlic seedling stage root diseases is as follows:

disease grading 0 grade:

the above-ground performance: the leaves are fresh green and have no pathological features. Root expression: the garlic disk has luxuriant hair roots and no pathological features.

Disease grading 1 grade:

the above-ground performance: only 1 leaf appeared slightly green, no more than 25% wilted, and the stem base was slightly soft. Root expression: the garlic sprout grows well, the coverage area of the water-soaked scab is less than 15 percent, and the number of garlic sprouts is not obviously reduced.

Disease classification 3 grade:

the above-ground performance: 1-2 leaves are yellow, no more than 25% and less than 75% of plants will wither, and the base of the stem is soaked in water. Root expression: the area of the water-soaked scab is more than or equal to 15 percent and less than 50 percent, the roots are short and the hairy roots are reduced.

Disease grading 5 grades:

the above-ground performance: the leaves become yellow in 2-3 leaves, the plants are short and wilted by over 75 percent, and the base parts of the stems are soft and brown and soaked in water. Root expression: the area of the water-soaked disease spots is more than 50 percent, the roots are short and fragile and easy to break, the garlic disk is light brown, and the rooting amount is obviously reduced.

Disease grading, grade 7:

the above-ground performance: all leaves become withered and yellow, all plants are wilted and dwarf, and the stem base is seriously rotted. Root expression: the water stain-like disease spots cover the whole root system, and the garlic disk does not have root.

The formula for calculating the onset of disease is as follows:

disease index (Σ [ (number of disease stages × number of diseased plants) ]/(total number of investigation × highest stage) × 100 (zhangbo, 2008, preliminary study of rhizopus rot of garlic in shandong, university of agriculture, xinjiang).

The control effect is (control morbidity index-treatment morbidity index)/control morbidity index multiplied by 100% (Zhangbo, 2008, preliminary study on the root rot of garlic pythium in Shandong province, Xinjiang university of agriculture).

The result shows that the disease-susceptible symptoms of the garlic begin to appear after the single inoculation of pathogenic bacteria for about 7 days, the garlic initially shows that the garlic gradually grows weaker, the root growing amount is less or no root grows, the leaves on the overground part gradually wither and yellow, finally the root shows a rotten state and moves from the root tip part to the root base part, the rotten part of the root is firstly changed from red to a colorless hollow state, the garlic red root rot symptom consistent with the pathogenicity determination phenomenon of the echinococcus terrestris (Setophorridis) H5 is shown, and the disease index is as high as 88.6%; the inoculation of antagonistic fungus Talaromyces liani DS59-19F has obvious prevention and control effect compared with a pathogenic bacteria control group, although the growth vigor of the control group is different compared with that of a clear water control group, most of the roots of plants are healthy, the disease index is 36.2%, most of the roots of the plants show 3-grade diseases, the control is more than 5 grades and more, the prevention and control effect reaches 59.1%, and a greenhouse water culture effect graph is shown in figure 4.

Example 4 growth promotion verification of Talaromyces liani DS59-19F for greenhouse cultivation

Respectively inoculating Talaromyces liani DS59-19F and positive control industrial bacteria Trichoderma harzianum ACCC 30371 (from China agricultural microbial strain collection management center) into PD culture medium, and culturing at 180r/min at 28 deg.C for 5 days in shaking table. Preparation of Talaromyces liani DS59-19F hypha suspension with a concentration of 1X 10 using PD Medium⁸cfu/mL for use. The garlic is sterilized and planted in nutrient medium, 3 granules per pot, and placed in a greenhouse, watered every day without using fertilizer. When the garlic seedlings grow to about 10cm, the prepared hypha suspension is poured around the garlic, 50mL of the hypha suspension is poured in each pot, and pouring clear water is used as a 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 DS59-19F has no obvious difference with a negative control group, after 15 days, the negative control group without the fungicide gradually has a leaf drop phenomenon, the later stage has a yellow leaf and a withered leaf phenomenon, the irrigating factory fungus Trichoderma harzianum ACCC 30371 positive control group has no obvious leaf drop phenomenon, but the plant is relatively short and small, and the later stage has a leaf drop phenomenon. As embodied in fig. 5. According to actual measurement, the plant inoculated with Talaromyces liani DS59-19F is obviously higher than a negative control group (P <0.05) in plant height, stem thickness and plant fresh weight indexes, which shows that the fungus Talaromyces liani DS59-19F has obvious growth promotion effect on garlic seedlings in the 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 DS59-19F group:

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

The numerical values of the plant height, stem thickness and fresh weight of the plant in each of the above groups are expressed as "mean. + -. standard deviation".

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> fungus strain with functions of antagonizing garlic root rot pathogenic bacteria and promoting growth

<130> GNCLN212792

<160> 9

<170> PatentIn version 3.5

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cgccgaagcg cgctgtgaac cctgatgaag atgggctgtc tgagtactat gaaaattgtc 180

aaaactttca acaatggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga 240

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cctggcattc cggggggcat gcctgtccga gcgtcatttc tgccctcaag cacggcttgt 360

gtgttgggtg tggtcccccc ggggacctgc ccgaaaggca gcggcgacgt ccgtctggtc 420

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caaaatgaag gacaccgact ccgaggaaga gatccgcgag gcattcaagg tgtttgaccg 420

tgacaacaat ggattcatct ctgctgccga attgcgccac gttatgacct cgattggcga 480

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Claims

Talaromyces liani, the strain number of which is DS59-19F, and the registration number of the strain number of the strain of Talaromyces liani of the strain.
2. A culture of Talaromyces liani according to claim 1, which is obtained by culturing Talaromyces liani according to claim 1 in a fungal culture medium.
3. The microbial inoculum is characterized in that: the microbial inoculum contains Talaromyces liani of claim 1, a metabolite of Talaromyces liani of claim 1, and/or a culture of claim 2.
4. The microbial inoculum of claim 3, 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.
5. The microbial inoculum according to claim 4, characterized in that: the pathogenic bacteria inhibitor has an inhibiting effect on garlic root rot pathogenic bacteria; the disease is garlic root rot.
6. The microbial inoculum of claim 5, wherein: the pathogenic bacteria inhibitor has an inhibiting effect on all or part of the following garlic root rot pathogenic bacteria: fusarium oxysporum, Fusarium fragrans, Fusarium angustifolium, Fusarium solani, and Sphaerotheca terrestris.
7. The microbial inoculum according to claim 4, characterized in that: the plant is any one of the following plants:

p1) garlic;

p2) plants of the allium genus;

p3) plants of the liliaceae family.
8. Use of any of the Talaromyces liani of claim 1, a metabolite of Talaromyces liani of claim 1 and/or a culture of claim 2:

1) inhibiting pathogenic bacteria;

2) preparing a pathogenic bacteria inhibitor;

3) inhibiting diseases;

4) preparing a disease inhibitor.
9. Use of any of Talaromyces liani as defined in claim 1, a metabolite of Talaromyces liani as defined in claim 1, a culture as defined in claim 2 and/or a bacterial agent as defined in any of claims 3-6, for any of the following:

A1) preparing a product for promoting plant growth;

A2) promoting the growth of plants;

A3) preparing a product for increasing plant yield;

A4) the yield of the plant is improved.
10. Use according to claim 9, characterized in that: the plant is any one of the following plants:

p1) garlic;

p2) plants of the allium genus;

p3) plants of the liliaceae family.