CN111440746B

CN111440746B - Ericerus pela capable of inhibiting fusarium pathogenic bacteria and application thereof

Info

Publication number: CN111440746B
Application number: CN202010336119.1A
Authority: CN
Inventors: 杨涛; 王治业; 赵疆; 马永贵; 谢惠春; 杨晖; 巩晓芳; 方彦昊; 赵志敏
Original assignee: Qinghai Normal University; Institute of Biology of Gansu Academy of Sciences
Current assignee: Qinghai Normal University; Institute of Biology of Gansu Academy of Sciences
Priority date: 2020-04-25
Filing date: 2020-04-25
Publication date: 2021-02-19
Anticipated expiration: 2040-04-25
Also published as: CN111440746A

Abstract

The invention relates to the technical field of microorganisms and agriculture, in particular to a lecanicillium kuwanense strain capable of inhibiting fusarium pathogenic bacteria and application thereof. The lecanicillium kurosou BMDBLJJ-1 is preserved in the China general microbiological culture collection center in 2019, 10 and 11 days, and the preservation number is CGMCC No.18585, and the lecanicillium kurosou BMDBLJJ-1 can inhibit fusarium pathogenic bacteria, remarkably reduce the incidence of root rot and pockmark of fritillaria and angelica caused by fusarium pathogenic bacteria infection, improve the green turning rate of fritillaria, and can be used for preventing and treating various plant diseases caused by the fusarium pathogenic bacteria.

Description

Ericerus pela capable of inhibiting fusarium pathogenic bacteria and application thereof

Technical Field

The invention relates to the technical field of microorganisms and agriculture, in particular to a lecanicillium kuwanense strain capable of inhibiting fusarium pathogenic bacteria and application thereof.

Background

Fusarium (Fusarium Link exFr), also known as Fusarium, belongs to deuteromycotina, order hypocrea, taxonomically in its asexual period. The sexual stage is the subdivision Ascomycotina, and the sexual state is usually Gibberella. Fusarium is a kind of fungi distributed worldwide, it can not only overwinter in soil, but also infect many economic crops, cause many diseases such as root rot, stem base rot, flower rot and ear rot of plants, the main diseases are manifested as rot, blight and numb mouth disease, host plants are various, including medicinal plants, economic crops, etc., it mainly causes crop wilting death, affects yield and quality, it is one of the most difficult important diseases to prevent and treat in production. The rot disease is caused by various pathogenic bacteria of fusarium, including root rot, stem rot, etc., and the root rot is a soil-borne disease and is mainly transmitted by moisture in soil, underground insects and nematodes. The leprosy is caused by mixed infection of fusarium fungi and insect pests, when local root-knot nematodes and pests are harmed, the local root-knot nematodes and the pests bite and cause wounds, and pathogenic bacteria carried by the nematodes and the soil pests and pathogenic bacteria in soil further infect the wounds to cause morbidity. At present, the control of plant root rot and rough mouth disease is mainly realized by chemical drugs, such as copper ammoniate, metalaxyl, carbendazim, ethylicin, copper humate and the like, which are poured to the roots of plants to control the occurrence of the root rot, but the drugs cause environmental pollution and drug residues. Biological control is a hot spot of research at present, however, the skilled person in the art has not developed an effective biological drug for controlling the root rot caused by fusarium pathogenic bacteria, and aiming at the technical problems, the invention aims to find a biological drug capable of effectively controlling the plant root rot.

Lecanicillium lecanii (Lecanicilliumpsalliotae) is a biocontrol fungus, Gams, of the phylum Ascomycota, family Cordyceps&Zare is named as Ericerus pela according to morphological characteristics and ITS sequence comparison and analysis, and the Ericerus pela is separated from the epiphyllum. The strain is cultured on PDA plate, the front side of the colony is white cotton-shaped, and the back side is redOr a purple-red color, often with the pigment diffusing into the agar. Conidiophores are undeveloped and similar to vegetative hyphae, phialides are grown on the conidiophores and are single-grown, double-grown or 3-4-root recurrent, fusarium type large conidia perpendicular to the phialides are formed at the tops of the phialides, and the fusarium type large conidia are bent and generally have sharp tail ends; the mycelium has a septum and can generate oval or elliptical microconidia; the lecanicillium kummense can produce pink or purple red pigment which is considered as a slightly toxic substance and is named as oosporin, and the oosporin has certain toxic effect on nematodes and aphids, for example, the lecanicillium kummense strain (CN102417886B) disclosed in the invention patent is lecanicillium kummense strain, with the preservation number: CGMCC NO. 5329. The strain can colonize eggs, 2-instar larvae and female worms of the root-knot nematodes in different living stages. The invention discloses an insecticide composition combining lecanicillium kurtsonii and matrine and application thereof in aphid control (CN 201910139559.5). The dosage ratio of matrine to lecanicillium kurtsonii in the insecticide composition is carried out according to the following conditions: the final concentration of the matrine is 0.39mg/L to 6.25mg/L, and the concentration of the lecanicillium kusnezoffii is 0.5 multiplied by 10⁷spore/mL-1.5X 10⁷spore/mL; and particularly discloses application of the compound in aphid control, and the compound can be used for controlling plant diseases caused by nematodes. The invention unexpectedly discovers a lecanicillium kuwana BMDBLJJ-1 strain which has different characteristics from the lecanicillium kuwana which is disclosed in the prior art and can obviously inhibit the growth of pathogenic bacteria of fusarium.

Disclosure of Invention

In view of the above technical problems, an object of the present invention is to provide a lecanicillium alcaligenes (lecanicillium allootae) BMDBLJJ-1 strain, which is deposited in the china general microbiological culture collection center at 10 and 11 months in 2019 with the preservation number of CGMCC No.18585, the preservation address: the western road No.1 Hospital No. 3, Beijing, Chaoyang, is in survival state, and the telephone: 010-: 010-64807288.

The Lecanicillium lecanii BMDBLJJ-1 has an ITS sequence shown in SEQ ID NO. 1.

The lecanicillium kuwana BMDBLJJ-1 is applied to inhibiting fusarium pathogenic bacteria.

Preferably, the fusarium pathogenic bacteria are fusarium oxysporum or fusarium solani.

The lecanicillium kuwana BMDBLJJ-1 is applied to the prevention and treatment of plant diseases caused by fusarium pathogenic bacteria.

Preferably, the plant is a medicinal plant, and the disease is one or more of rot, blight and numb mouth disease caused by fusarium pathogenic bacteria.

Preferably, the disease is fritillaria root rot, angelica root rot or angelica nummularia.

A pesticide for preventing and treating plant diseases caused by fusarium pathogenic bacteria contains the lecanicillium kuwana BMDBLJJ-1.

Preferably, the pesticide contains the lecanicillium kuwana BMDBLJJ-1 spore with the number of 10⁶～10⁸/ml。

The invention has the beneficial effects that: the invention provides a new lecanicillium kummensen BMDBLJJ-1, which has nitrogen fixing capacity and strong secretion capacity, and the metabonomics of the secretion is detected by an LC-MS means, so that the lecanicillium kuchenense has the functions of killing insects, inhibiting and killing fungi, inhibiting and killing bacteria, inhibiting weeds, regulating plant growth, secreting allelochemicals, promoting symbiosis of plants and the like, and a flat plate antagonism experiment proves that the lecanicillium kuchenense BMDBLJJ-1 has antagonism on pathogenic bacteria of fusarium, so that the incidence rate of root rot and leprosy of fritillaria and angelica is obviously reduced, the green returning rate of fritillaria is improved, and the lecanicillium kunsen can be used for preventing and treating plant diseases caused by pathogenic bacteria of fusarium.

Drawings

FIG. 1 the front side of Ericerus pela bacterial colony in PDA solid medium

FIG. 2 the back of Ericerus pela bacterial colony in PDA solid medium

FIG. 3 the secretion of pink pigment from Lecanicillium lecanii in PDA liquid medium

FIG. 4A liquid pearl secreted by Lecanicillium lecanii

FIG. 5 phylogenetic tree of Lecanicillium lecanii

FIG. 6 is a schematic diagram of nitrogen fixation by Lecanicillium lecanii

FIG. 7 Total ion flow chromatogram (ESI +) of liquid bead sample

FIG. 8 liquid bead sample Total ion flow chromatogram (ESI-)

FIG. 9 is a schematic diagram showing the antagonism of Ericerus pela and Fusarium solani plates

FIG. 10 is a schematic diagram showing the antagonism of Lecanicillium chrysosporium and Fusarium oxysporum plates

FIG. 11 shows the control of root rot of potted fritillary

Detailed Description

The present invention will be further described with reference to the following examples, but the scope of the present invention is not limited to the following examples, and modifications or substitutions of the methods, steps or conditions of the present invention can be made without departing from the spirit and substance of the present invention.

The fritillaria kansuensis used in the following experiments was collected from Min county over 2800 m above, and the angelica sinensis was collected from Min county over 2500 m above. The reagents and culture media used in the test are all chemically pure. The reagent related to metabonomics determination is chromatographically pure.

Medium composition and noun explanation:

a tiger red culture medium: 5g/L of peptone, 10g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.03g/L of Bengal, 0.1g/L of chloramphenicol, 15g/L of agar and 7.2 +/-0.2 of final pH.

Liquid PDA culture medium: potato 200g/L, glucose 20g/L, natural pH. Solid added agar 15 g/L.

An abb culture medium: 0.2g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L sodium chloride, 5.0g/L calcium carbonate, 10.0g/L mannitol, 0.1g/L calcium sulfate, 15.0g/L agar and pH value of 7.0.

Liquid LB medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 7.4 of pH value. Solid added agar 15 g/L.

SDS (sodium dodecyl sulfate): also called sodium dodecyl sulfate, is an anionic detergent, can lyse cells under the condition of high temperature, make chromosome segregation, protein denaturation, SDS combines into the complex with protein and glycan at the same time, release nucleic acid; the solubility of the SDS-protein compound is smaller by increasing the salt concentration and reducing the temperature, so that the protein and polysaccharide impurity precipitation is more complete, and the precipitate is removed after centrifugation; the DNA in the supernatant was extracted with phenol/chloroform, and after repeated extraction, the DNA in the aqueous phase was precipitated with ethanol.

Medicinal plants: refers to a plant used for preventing and curing diseases in medicine. All or a part of the plants are used for medicine or as raw materials in the pharmaceutical industry. In a broad sense, plant sources useful as nutrients, certain hobbies, flavorings, color additives, and pesticides and veterinary drugs can be included.

Example one Strain isolation

Cleaning harvested healthy Gansu fritillary and angelica respectively for 10min by running water, sucking surface water by using filter paper, cutting off roots of fritillary and hairy roots of angelica respectively, washing the fritillary and hairy roots of angelica for 3-4 times by using sterile distilled water, soaking the fritillary and hairy roots of angelica for 30s by using 75% ethanol, washing the fritillary and hairy roots of angelica for 2-3 times by using sterile water, sterilizing the fritillary and hairy roots by using 0.2% mercuric chloride for 10min, washing the fritillary and hairy roots of angelica for 3-4 times by using the sterile water, and finally coating 100 mu l of the washed sterile water on a tiger red solid culture medium as a control.

Respectively sucking surface water of the explant of the fritillaria and the angelica after surface disinfection treatment by sterilized filter paper, cutting the explant into about 1cm by using a sterilization knife, putting 1g of the explant into a mortar subjected to high-temperature dry heat sterilization at 200 ℃, adding 10ml of sterilized physiological saline, and fully grinding.

The mass fraction of the stock solution after grinding is 10^-1Sequentially sucking 1mL of the mixture, adding the mixture into sterilized 9mL of physiological saline, and diluting to obtain 10 mass fractions^-2、10^-3、10^-4The solution of (1). Sucking 100 μ l of the above liquid, repeating each concentration gradient for 3 times, spreading on solid tiger red plate, culturing at 28 deg.C in dark for about 15 days, and growing different fungi.

Carefully picking hyphae from the sides of different colonies, streaking and purifying on a solid PDA plate for 3-5 times, inoculating the purified strain to a PDA inclined plane to obtain the purified strain, and preserving at a low temperature of 4 ℃. Picking up a little hypha from different strains, preparing water slices, and observing microscopically. One of them bacterial colony form is unique, and the bacterial colony front is white, and the bacterial colony back is pink, secretes the haematochrome to there are a lot of liquid drops on the bacterial colony surface secretion, picks the bacterial colony and carries out follow-up experiment. The specific form is shown in figures 1-4.

EXAMPLE two Strain identification

Respectively inoculating different strains obtained in the first embodiment into a liquid PDA culture medium, culturing at 28 ℃, 200rpm for 7-10 days, centrifuging at 4 ℃, 5000rpm to collect thalli, fully crushing by a tissue crusher, extracting genome DNA by an SDS method, and performing PCR amplification after passing electrophoretic detection. The primers are as follows:

ITS1：5'-TCCGTAGGTGAACCTGCGG-3'

ITS4：5'-TCCTCCGCTTATTGATATGC-3'

the PCR reaction system is 50 μ l:5

Buffer(Mg²⁺plus) 10. mu.l, forward and reverse primers 2. mu.l each, dNTP mix (2.5 mM each) 4. mu.l,

HS DNA Polymerase (2.5 μm) 0.5. mu.l, DNA template 1. mu.l, ddH₂O 31.5μl。

PCR amplification conditions: denaturation at 98 ℃ for 10 sec; annealing at 56 ℃ for 15sec, extension at 72 ℃ for 1min 30sec, 30 cycles, and extension at 72 ℃ for 5 min. The amplified product is sent to Beijing Optimalaceae new biotechnology limited for sequencing. Sequencing results were aligned in the NCBI database blast, and MEGA7 constructed a phylogenetic tree.

The strains separated from the fritillaria and the angelica are identified as the same strain by PCR, the length of an ITS sequence is 575bp, the sequence table is shown as SEQ ID NO.1, and the homology with the Ericerus pela strain CBS344.37 is 99.28%. Therefore, the strain isolated in the experiment is Ericerus pela (Lecanicilliumpsilotae), is named as BMDBLJJ-1, is preserved in the China general microbiological culture collection center in 2019, 10 and 11 months, and has the preservation number of CGMCC No. 18585.

The construction of the clade using MEGA7 with the different species of Lecanicillium as the inner population and Verticillium lecanii, Trichoderma harzianum, and Gliocladium roseum as the outer population is shown in FIG. 5. As can be seen from FIG. 5, the isolated strain tested (number 1) was Ericerus pela, which has a closest relationship to the Ericerus aphanocadii species, followed by Verticillium lecanii, and followed by the Ericerus acerosa species. Trichoderma harzianum has a close relationship with Gliocladium roseum, but has a far relationship with other fungi.

Example qualitative determination of Nitrogen fixation Activity of Lecanicillium tricolor BMDBLJJ-1

Preparing an Arthropoda Beauda culture medium, adding 5ml/L of 0.5% Congo red, sterilizing, preparing into a flat plate for later use, selecting a small amount of hyphae from the edge of the preserved Erysipelothrix Geranii BMDBLJJ-1 colony, inoculating into the Arthropoda Beauda culture medium, culturing at 28 ℃ in the dark for 10 days, and observing the growth condition of the colony.

Congo red is brownish red powder, is yellow red when dissolved in water, is orange when dissolved in alcohol, is used as an acid-base indicator, has the color change range of 3.5 to 5.2, is red in an alkaline state and is bluish purple in an acid state, and is a red ring when the nitrogen fixation of the lecanicillium kurosou BMDBLJJ-1 is in an alkaline state. The size and the color of the ring can reflect the strength of the nitrogen fixation capacity to a certain extent.

The experimental result is shown in FIG. 6, the colony on the nitrogen-free Allium cepa culture medium is clearly visible, and a red circle is formed, which indicates that the nitrogen fixing ability of Ericerus gasseri BMDBLJJ-1 is strong.

Example Ericerus pela BMDBLJJ-1 secretion LC-MS metabolome assay

A sterile needle tube is used for sucking liquid drops secreted by the lecanicillium kuwana strain BMDBLJJ-1, the liquid drops are sent to Nanjing Jisi Huiyuan biotechnology limited company for measuring LC-MS metabonomics, and the function or potential function of related compounds is searched by searching documents.

1. Metabolite extraction

Putting 5mL of sample into a 10mL centrifuge tube, and freeze-drying in a freeze dryer for 12 h; add 500. mu.L of 80% methanol to the lyophilized sample and vortex for 1 min; performing ultrasonic treatment at 4 ℃ for 30 min; standing at-20 deg.C for 1 h; centrifuging at 12000rpm at 4 deg.C for 15 min; 200. mu.L of the supernatant was removed, 5. mu.L of internal standard (2.8mg/mL, dichlorophenylalanine) was added, and transferred to a sample vial for LC-MS detection and analysis.

2. Detection on machine

An instrument analysis platform: LC-MS (Thermo, Ultimate 3000LC, QOxctive); a chromatographic column: c₁₈Column (Hypergold C18(100X2.1mm 1.9 μm)); the chromatographic separation conditions are as follows: the column temperature was 40 ℃; the flow rate is 0.3 mL/min; mobile phase composition A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid; the sample injection amount is 4 mu L, and the temperature of an automatic sample injector is 4 ℃;

the mobile phase gradient elution procedure is shown in table:

TABLE 1 liquid chromatography mobile phase conditions

Mass spectrum detection parameters:

the positive mode is as follows: the temperature of the heater is 300 ℃; flow rate of sheath gas: 45 arb; flow rate of auxiliary gas: 15 arb; tail gas flow rate: 1 arb; electrospray voltage: 3.0 KV; capillary temperature: 350 ℃; S-Lens RF Level, 30%.

Negative mode: the temperature of the heater is 300 ℃; flow rate of sheath gas: 45 arb; flow rate of auxiliary gas: 15 arb; tail gas flow rate: 1 arb; electrospray voltage: 3.2 KV; capillary temperature: 350 ℃; S-Lens RF Level, 60%.

Scanning mode: a primary Full Scan (Full Scan, m/z 70-1050) and a data-dependent secondary mass spectrometry Scan (dd-MS2, TopN 10); resolution ratio: 70,000 (primary mass spectrum) &17, 500 (secondary mass spectrum). Collision mode: high energy collision dissociation (HCD).

3. Data processing

LC/MS detection data is extracted and preprocessed by using Compound reader software (Thermo company), and is arranged into a two-dimensional data matrix form, wherein the two-dimensional data matrix form comprises information such as Retention Time (RT), Molecular Weight (Molecular Weight), observed quantity (sample name), peak intensity and the like.

4. Analysis of results

(ESI +) represents a positive ion detection mode, i.e., the mass analyzer scans only positive charge ions and filters out negative charge ions during detection, thereby obtaining information of the positive charge ions; (ESI-) represents a negative ion detection mode, i.e. the mass analyzer scans only negative charged ions and filters out positive charged ions during detection, thereby obtaining information of the negative charged ions. Fig. 7 and 8 are chromatograms of positive and negative ion flows, respectively. Table 2 shows the compound name, molecular weight, retention time, peak area detected by positive ion flow chromatography. Table 3 shows the name of the compound, molecular weight, retention time, peak area detected by anion flow chromatography.

TABLE 2 name of compound, molecular weight, retention time, peak area detected by positive ion current chromatography

TABLE 3 name of compound detected by anion flow chromatography, molecular weight, retention time, peak area

According to the literature, the functional classification of the compounds detected is summarized as follows:

(1) colorless transparent droplets are composed mainly of the following classes of compounds: benzenoids (benzoic acid and derivatives, phenylacetic acid and derivatives, benzenesulfonic acid and derivatives, anisoles, aromatics, benzoyl derivatives, toluene), lipid and lipoid molecules (fatty acids and conjugates, fatty amides, vitamin D and derivatives, fatty acid esters, sesquiterpenes), aromatic heterocycles (picolinic acid and derivatives), organic acids and derivatives (tricarboxylic acids and derivatives, amino acids, polypeptides, and the like), phenylpropionic acid and polyketones (isoflavanquinones), organic heterocycles (purines and purine derivatives, indoles and derivatives, morpholines, pyrones and derivatives, caprolactams, tryptamines and derivatives, pyridoxamines, imidazoles), organic nitrogen compounds (amines, alkanolamines, primary amines, cholines), alkaloids and derivatives, nucleosides, nucleic acids and analogs, Organic oxygen compounds (aldehydes).

(2) The substances with the functions of inhibiting and killing bacteria and potential in the liquid drops are as follows: 2-Hydroxyaproic acid, 4-oxo-4- [ (pyridine-4-ylmethyl) amino ] but-2-enoic acid, isoproxyl 4-hydroxybenzoatecanamine, Citric acid, Bromic acid, Azelaic acid, Chloramphenicol, sulfomethoxine, 2- [ (1S, 2S, 4aR, 8aS) -1-hydroxy-4 a-methyl-8-methyl-naphthalene-2-yl ] prop-2-enoic acid, Butylobe, methyl 3- (methyalamo) -2- [ (5-methyl-3-isopropylyl) carbonyl ] -2-enoic acid, and the like.

(3) The substances with the functions of inhibiting and killing fungi and potential in the liquid drops are as follows: benzoic acid, 2- [ (E) - (3-Nitro benzylidene) amino ] Benzoic acid, Butylparaben, Tridemorph, (3, 4, 5-trihydroxy-6- { [4- (2, 6, 6-trimethy-4-oxolox-2-en-1-yl) butan-2-yl ] oxy } oxan-2-yl) methyl 3, 4, 5-trihydroxybenzoate, 3, 5-di-tert-Butyl-4-hydroxybenzoic acid, pyalol, Docosamide, etc.

(4) The insect inhibiting and killing effect and potential substances in the liquid drop are as follows: citraconic acid, Lauric acid, 2- (8-Hydroxy-4a, 8-dimethylhydroxy-2-naphthol) acrylic acid, Caprolactam, 7-Hydroxysubunit, Tolycaine, Docosanamide, 3-Hydroxy Picolinic acid, 8-Hydroxyquinine, Kojic acid, Dibutyl phthalate, Picolinic acid, 12-Amino docosanoic acid, tert-Butyl N- [1- (aminocarbonyl) -3-methyxyl ] carbamate, (2E) -3- (4-Acetoxy-2, 3-dihydroxy-2, 5, 5, 8 a-tetramethyldehydro-1-naphthol) and the like.

(5) The expelling substances in the droplets that attract the probiotic bacteria are: d- (+) -Malic acid.

(6) The weed inhibiting effect and potential substances in the liquid drop are as follows: 3-Hydroxy-2-methylpyridine, 3-Hydroxy Picolinic acid, Imazethapyr, and the like.

(7) The substances for promoting plant growth and potential in the liquid drops are as follows: 3-Methylglutaric acid, 5-Hydroxyindole-3-acetic acid, octadec-9-ynoic acid, Trigonelline.

(8) The pesticide (sterilization inhibiting and disinsection inhibiting) intermediates in the liquid drops comprise: N-Allyl-2, 2, 2-trifluoroacetamide, 3-Chlorobenzoic acid, N1- (9, 10-dihydrophenanthren-2-yl) acetamide, 4-Nitrobenzoic acid, Diethyl dialialonate, Pyroglenol, Triphenylsphasphophenol oxide, N-Acetyl sarcosine, Dichloroacetamide, 6-Methoxyquinoline, 4-buthylamine, and the like.

Example Lecanicillium lecanii BMDBLJJ-1 plate antagonism assay

1. Experimental groups and strain sources

Fusarium oxysporum: pathogenic bacteria isolated by the laboratory technician from fritillaria and angelica that have rot, blight; fusarium oxysporum described in this example is a common pathogenic bacterium and can also be obtained from strain depositories or purchased from reagent companies by those skilled in the art.

Fusarium solani: pathogenic bacteria isolated by the laboratory technician from fritillaria and angelica that have rot, blight; fusarium oxysporum described in this example is a common pathogenic bacterium and can also be obtained from strain depositories or purchased from reagent companies by those skilled in the art.

2. Procedure of experiment

Respectively inoculating fusarium oxysporum and fusarium solani to the center of a PDA solid flat plate, inoculating the lecanicillium kuwanense BMDBLJJ-1 to the periphery of pathogenic bacteria at a distance of 2cm, culturing at 28 ℃ for 5-7 days, observing the growth of bacterial colonies and the conditions of antibacterial zones, measuring the radius of the antibacterial zones and the radius of antagonistic bacteria, and calculating the antagonistic index.

Antagonism index ═ radius of zone of inhibition-radius of antagonistic bacteria)/radius of antagonistic bacteria

The radius of the inhibition zone is equal to the distance from the center of the antagonistic bacteria colony to the edge of the hypha of the pathogenic bacteria

3. Results of the experiment

The antagonistic results of the strains are shown in figures 9 and 10, and it can be seen from the figures that the bacterial colony of the lecanicillium kuwanense BMDBLJJ-1 is dense, circular to elliptical, the contact surface with pathogenic bacteria is in a crescent shape, and the bacteriostatic action is obvious.

The antagonism index is calculated according to the formula, the antagonism index of the lecanicillium kurosou BMDBLJJ-1 to the fusarium solani is (133 +/-6.7)%, and the antagonism index to the fusarium oxysporum is (92 +/-5.3)%. The results show that the Ericerus pela BMDBLJJ-1 strain has antagonistic action on pathogenic bacteria of fusarium.

Example application of Lecanicillium hexacercosporum BMDBLJJ-1 in prevention and treatment of root rot of potted fritillary

In order to evaluate the prevention and treatment effect of the lecanicillium kummenseng BMDBLJJ-1 on the root rot of the fritillaria caused by fusarium pathogenic bacteria, the embodiment firstly carries out an evaluation experiment by taking potted fritillaria as an experimental object, the embodiment is divided into an experimental group, a positive control group and a blank group, the experimental group is irrigated with the lecanicillium kuchensinensis BMDBLJJ-1, the positive control group is irrigated with the bacillus amyloliquefaciens, and the blank group is irrigated with sterilized water. The specific experimental process is as follows:

the mixed matrix used for cultivation in this example is obtained by uniformly mixing turfy soil, rice husk charcoal and vermiculite according to a volume ratio of 3:1: 1.

The spore number of the Lecanicillium lecanii BMDBLJJ-1 bacterial liquid irrigated by the embodiment is 10⁶～10⁷/ml。

Experimental groups: soaking the fritillaria bulb with white, smooth and disease-free surface in 0.2% mercuric chloride, sterilizing the surface for 20 minutes, and cleaning with clear water. Culturing the cleaned Bulbus Fritillariae Cirrhosae seed balls in sterilized mixed matrix at room temperature under relative humidity of 60%, and repeating for 3 times for 50 Bulbus Fritillariae Cirrhosae seed balls per group. Diluting the bacterial liquid of Ericerus pela BMDBLJJ-1 by 100 times, pouring the diluted bacterial liquid, pouring the bacterial liquid of Fusarium oxysporum 5 days later, and counting the number of spores>10⁸Ml, pouring Fusarium solani bacterial liquid after 10 days, the number of spores>10⁸Each of the above Fusarium oxysporum and Fusarium solani was diluted 100-fold at the time of pouring. Disease conditions were counted after 30 days, and statistical analysis was performed by the SPSS 22 software.

Positive control group: soaking the fritillaria bulb with white, smooth and disease-free surface in 0.2% mercuric chloride for 20 minutes, and cleaning with clear water. Culturing the cleaned Bulbus Fritillariae Cirrhosae seed bulbs in sterilized mixed matrix at room temperature under relative humidity of 60%, and repeating for 3 times for 50 Bulbus Fritillariae Cirrhosae seed bulbs per group. Diluting the bacillus amyloliquefaciens bacterial liquid by 100 times (the bacterial number is 10)⁶～ 10⁷Perml), and after 5 days, fusarium oxysporum bacterial liquid (number of spores) is poured in>10⁸Ml), after 10 days fusarium solani bacterial liquid (spore number) is poured in>10⁸/ml), the above Fusarium oxysporum and Fusarium solani were both diluted 100-fold at the time of pouring. Disease conditions were counted after 30 days and analyzed statistically by the SPSS 22 software.

Blank group: soaking the fritillaria bulb with white, smooth and disease-free surface in 0.2% mercuric chloride, sterilizing the surface for 20 minutes, and cleaning with clear water. Culturing the cleaned Bulbus Fritillariae Cirrhosae seed balls in sterilized mixed matrix at room temperature under relative humidity of 60%, and repeating for 3 times for 50 Bulbus Fritillariae Cirrhosae seed balls per group. Pouring sterilized water with volume equal to that of experimental Ericerus pela BMDBLJJ-1 culture solution, and pouring Fusarium oxysporum bacterial solution (number of spores) after 5 days>10⁸Ml), after 10 days fusarium solani bacterial liquid (spore number) is poured in>10⁸/ml), the above Fusarium oxysporum and Fusarium solani were both diluted 100-fold at the time of pouring. Disease conditions were counted after 30 days and analyzed statistically by the SPSS 22 software.

Taking the disease symptoms of yellowing, yellow spots and rot of the surfaces of the fritillary bulbs as standards, counting the disease condition of each group of fritillary bulbs, wherein the statistical result is shown in fig. 11, the disease rate of a blank group is (90 +/-5.3)%, the disease rate of a positive control group is (40 +/-3.4)%, and the disease rate of an experimental group is (20 +/-2.6)%; the positive control group treated by the bacillus amyloliquefaciens and the experimental group treated by the lecanicillium kuwanense BMDBLJJ-1 both obviously reduce the incidence rate of fritillaria root rot; compared with a positive control group, the experimental group treated by the lecanicillium kuwanense BMDBLJJ-1 obviously reduces the incidence rate of fritillaria root rot; therefore, the fritillaria treated by the cuphea canicola BMDBLJJ-1 irrigation has the effect of reducing the incidence rate of the fritillaria root rot, is superior to the control effect of bacillus amyloliquefaciens on the fritillaria root rot, and can be applied to control the root rot caused by fusarium pathogenic bacteria.

Example application of Lecanicillium septemlobus BMDBLJJ-1 in preventing and treating fritillary root rot

In order to further evaluate the control effect of lecanicillium kuwanense BMDBLJJ-1 on the root rot of fritillaria, the field transplanted fritillaria is taken as an experimental object in the embodiment, and the embodiment is divided into three groups, namely an experimental group, a blank control group and a traditional method control group. The experimental group is realized by pouring Lecanicillium lecanii BMDBLJJ-1 bacteria 3 times in the seedling cultivation stage of the fritillaria and coating the small stem of the fritillaria with Lecanicillium lecanii BMDBLJJ-1 bacteria liquid before transplanting; the fritillaria cultivation method of the blank control group is the same as that of the experimental group, but the bacterial liquid of lecanicillium kuwanense BMDBLJJ-1 is not poured; the control group is cultured by a traditional method, and the bacterial liquid of Ericerus pela BMDBLJJ-1 is not irrigated in the culturing process; the method comprises the following specific operation steps of counting the incidence rate and the green turning rate of the root rot of an experimental group, a blank control group and a traditional method control group:

the main differences between the simulated cultivation test base of county horse slope village (elevation 2600 m) in elm as the cultivation base of the Huating city horse gorge town (elevation 1800 m) in the embodiment are temperature difference caused by elevation, and similar factors influencing the occurrence of fritillaria diseases such as rainfall, rainfall period and the like. During cultivation, 70% of sunshade net is respectively arranged at the two bases, and the land is the fritillaria continuous cropping land.

The mixed substrate used for cultivation in this example is composed of peat soil for seedling: granular vermiculite: the rice husk carbon is mixed according to the volume ratio of 3:2: 1.

The number of spores of the Lecanicillium lecanii BMDBLJJ-1 bacterial liquid irrigated in this example was 10⁸/ml。

The incidence of diseases is as follows: counting in the middle and last ten days 4 months after seedling emergence;

green turning rate: in the middle and last ten days of the next 4 months (the incidence rate is counted in the next year), as the rainy season in Gansu is mainly concentrated in 7-9 months, although the fritillaria is fallen to seedlings, the fritillaria buried in the soil is easy to suffer from root rot, the green return rate in the next year is reduced, and the green return rate in the next year is counted.

Experimental groups:

(1) bulbus Fritillariae Cirrhosae seed dormancy breaking method

Collecting seeds of fritillaria kansuensis in 6 months in 1 year (2016), storing at 16 ℃ for 14h/8 ℃ for 10h in sand at variable temperature for 3 months until the embryo rate reaches above 0.6, and then storing at 2 ℃ for 2 months in sand at constant temperature until the embryo rate reaches above 0.8;

(2) seed sowing

Sterilizing the seeds after post-ripening for 10min with 0.2% mercuric chloride, washing with sterile water, sowing in sterilized mixed matrix, culturing in greenhouse condition of 1 month in 2 years (2017), and controlling the minimum temperature of the greenhouse to be more than 0 deg.C;

(3) inoculating, culturing, and storing at low temperature

The seed in the step (2) begins to emerge about 20 days after the seed is sown, the 1 st time after the emergence of the seed is watered with the bacterial liquid of lecanium kusnezoffii BMDBLJJ-1, the number of the spores is 10⁸Diluting by 100 times when the water-soluble micro-emulsion is used, pouring seedlings when the seedlings grow to 2017 for 5 months, and performing statistics to show that the bulblet size is 2-3 mm 2 ℃ and low-temperature storage is performed for 3 months;

(4) seeding the bulblet to harvest the bulblet

After 8 months of heatstroke in 2017, sowing the bulblets obtained in the step (3) into a mixed matrix, and culturing under a greenhouse condition with the highest temperature of the greenhouse<The seedling emergence is started about 20 days at the temperature of 30 ℃, the bacterial liquid of Ericerus pela BMDBLJJ-1 is irrigated for the 2 nd time after the seedling emergence, and the spore number of the bacterial liquid is 10⁸/ml, diluting 100 times, growing bulblet to 2017 for 12 months, and gradually reducing temperatureThe room temperature is up to the point that the fritillary is poured. At the moment, the size of the bulblet obtained by sowing is 3-4 mm, and the bulblet conforms to the size of the bulblet transplanted in the field;

(5) low-temperature storage and coating of small seed stems

Burying the microspecies obtained in the step (4) in a mixed matrix, irrigating and diluting 100 times of Ericerus pela BMDBLJJ-1 (3 rd time), and storing at 2 ℃ for 3 months. Taking out the small seed stems in the mixed matrix in 3 months in 3 years (2018), soaking the small seed stems in 100-time diluted Ericerus pela BMDBLJJ-1 bacterial liquid (4 th time) containing 0.05 percent of hydroxypropyl methyl cellulose for coating, taking out the small seed stems after soaking for 20min, drying the surface water in the shade, and using the small seed stems for field transplantation;

(6) transplanting in field

And respectively transplanting the small seed stems in a field in a simulated cultivation test base of a hillside country in Yuzhong county and a domestication cultivation test base of a strait town in Huating city. Transplanting 5000 seedlings in 3 rd year (2018) in the first 4 th month (pseudo-habitat cultivation base) and the last 3 th month (domesticated cultivation base), wherein the transplanting row spacing is 10cm, and the plant spacing is 5 cm. After seedling emergence, the incidence rate of root rot is counted in 4-5 months in 2018, and the green-turning rate is counted in 4-5 months in 2019.

Blank control group:

except that the fritillaria seeds are not watered with lecanicillium kummensen BMDBLJJ-1 (no water for 4 times), all cultivation methods of the blank control group are the same as those of the experimental group, the growth condition of the fritillaria is observed, the incidence rate of root rot is counted in 4-5 months in 2018, and the green return rate is counted in 4-5 months in 2019.

Control group of conventional method:

adopting a field direct seeding method to raise seedlings (different from methods of an experimental group and a blank control group), collecting seeds of fritillaria kansuensis in 1 year (2015 year) when half yellow fruits are not completely mature and cracked, selecting a slope field to raise ridges and sow the collected seeds, covering straws, pine needles and the like for preserving soil moisture, and seedling emergence at the bottom of 3 months in 2 years (2016 years) and seedling inversion at the bottom of 6 months; in the 3 rd year (2017), 3 months at the bottom of the seedling, 6 months at the bottom of the seedling, taking out the seedling in 10 months to harvest 2 years of small stems of fritillaria thunbergii, then respectively transplanting the stems in a cultivation test base of Ma Pop county in Yuzhong county and a cultivation test base of Maxia town domestication in Huating City, setting up 70% of sunshade nets on the two bases respectively, wherein the soil is a field for replanting fritillary bulbs, the transplanting row spacing is 10cm, the planting distance is 5cm, the seedlings emerge in 3 th to 4 th ten days after the 4 th year (2018) after the transplanting, the incidence rate of root rot is counted in 4 to 5 th days, and the green return rate is counted in 4 to 5 th days after the 2019 year.

TABLE 4 statistics of the growth of fritillaria under the same cultivation method

Growth conditions	Experimental group	Blank control group
			End of first life	2-3 mm in diameter	Diameter of 1-1.5 mm
End of second growth phase	The diameter is 3-4 mm	The diameter is 2-2.5 mm
			Emergence rate in the second growth period (%)	70±3.4*	20±1.8

Note: means significant difference in comparison between experimental and control groups

TABLE 5 growth of fritillaria with different cultivation methods and cultivation bases

Note:^#shows that the comparison between different cultivation bases has significant difference

Means significant difference in comparison between experimental and control groups

The growth conditions of the fritillaria under the same cultivation method are shown in table 4, the fritillaria seeds in the experimental group are sowed in the mixed matrix, the growth conditions are good, and no diseases occur; compared with the experimental group, the diameter and the emergence rate of the seedlings of the blank control group are lower than those of the experimental group, so that the lecanicillium kuchenense BMDBLJJ-1 provides nitrogen nutrition for the seedlings of the fritillaria kansuensis, promotes the growth, and provides guarantee for the development of the heart buds.

The growth conditions of the fritillaria in different cultivation methods and cultivation bases are shown in table 5, and compared with a control group in the traditional method, the incidence of diseases in an experimental group is lower, and the green turning rate is higher; compared with the Huating pavilion domestication cultivation base, the planting base in the elm is lower in incidence rate and higher in green turning rate.

In conclusion, the incidence rate of root rot of the fritillaria thunbergii irrigated with the lecanicillium kuwanii BMDBLJJ-1 bacterial liquid is remarkably reduced and the green returning rate is remarkably improved compared with the fritillaria thunbergii irrigated with no lecanicillium kuwanii BMDBLJJ-1, so that the lecanicillium kuwanii BMDBLJJ-1 has a protection effect on the root of the fritillaria thunbergii, the protection effect is not limited to laboratory conditions, the field planting also has the same effect, and the occurrence of the root rot of the fritillaria thunbergii can be prevented.

Example application of Lecanicillium octacelli BMDBLJJ-1 to control of root rot and pockmark disease of Angelica sinensis

In order to evaluate the prevention and treatment effects of Ericerus pela BMDBLJJ-1 on the root rot and the numb mouth disease of Angelica sinensis, the present example was divided into two groups, which were an experimental group and a control group. The experimental group is realized by three steps of irrigating lecanicillium kuwanense BMDBLJJ-1 bacterial liquid for 1 time in the seedling cultivation stage of the angelica sinensis, coating the seedlings with lecanicillium kuwanense BMDBLJJ-1 bacterial liquid before transplanting, and irrigating roots of lecanicillium kuwanense for 1 time after transplanting the seedlings in a field; the control group is not irrigated with lecanicillium kuwana BMDBLJJ-1 bacterial liquid; comparing the incidence rates of the root rot and the pockmark disease of the experimental group and the control group, and the specific operation steps are as follows:

note: because the root rot of angelica is concentrated in early spring seasons and the environmental conditions of dry cooling and wet cooling are easy to cause diseases, the disease occurrence condition of the root rot is counted in the middle of 6 months; the occurrence and the disease degree of the leprosy can not cause plant death, so the statistics of the disease degree of the leprosy is not carried out in the experiment, and only the disease condition is counted.

The mixed substrate used for cultivation in this example is seedling turfy soil: granular vermiculite: the rice husk charcoal is obtained by mixing the rice husk charcoal with the ratio of 3:2: 1.

The spore number of the Lecanicillium lecanii BMDBLJJ-1 bacterial liquid irrigated by the embodiment is 10⁸/ml。

Experimental groups:

1. seed sowing

Harvesting angelica seeds from the end of 10 months to the beginning of 11 months in the 1 st year, sterilizing by 0.2 percent mercuric chloride for 10min, washing by sterile water, sowing in a sterilized mixed matrix in 12 months, culturing under a greenhouse condition, controlling the lowest temperature of the greenhouse to be more than 0 ℃, starting seedling emergence in about 10 days, irrigating 100 times diluted lecanium kummersivum BMDBLJJ-1 bacterial solution for 1 time after seedling emergence, controlling the seedling stage to be 3-4 months according to the seedling growth potential, and gradually reducing the temperature in the middle and late 3 months in the 2 nd year to ensure that leaves are yellow and seedlings are lifted and transplanted; 2. seedling coating field planting

In the second year 3, seedlings are lifted at the bottom of month, the seedlings are soaked in 100-time diluted Ericerus pela BMDBLJJ-1 bacterial liquid containing 0.05 percent of hydroxypropyl methyl cellulose for coating, the seedlings are taken out after being soaked for 20min, the surface moisture is dried in the shade, the seedlings are transplanted in a field, the test field is located in Ten miles town (the altitude of 2200 m) in Min county, the transplanting field block is an angelica sinensis continuous cropping field, the transplanting row spacing is 20cm, the plant spacing is 10cm, and single-seedling hole planting is carried out to obtain 5000 seedlings in total;

3. irrigating roots after seedling emergence

The coated seedlings begin to germinate about 10 days after being planted, and when the seedlings grow to 3-4 leaves in the early 5 months of the 2 nd year, the bacterial liquid of the lecanicillium kurosporum BMDBLJJ-1 is diluted by 100 times and then the roots are irrigated.

4. Disease statistics

In 6 th month and middle day of 2 year, collecting root rot disease condition, collecting radix Angelicae sinensis at 11 th month of 2 year, collecting disease condition of rhizoma Gastrodiae disease, and collecting data without bolting radix Angelicae sinensis.

Control group:

and (3) directly seeding and raising seedlings in the field in the middle ten days of 6 months, controlling the seedling period to be about 3 and a half months, lifting the seedlings in 10 months, bundling and stacking the seedlings, transplanting the seedlings at the bottom of 3 months in the next year, and enabling the transplanting place and method to be the same as the experimental group. In 6 middle of month in 2 year, the incidence of root rot is counted, Chinese angelica is harvested at 11 months in 2 year, the incidence of rough mouth disease and root rot is counted, and Chinese angelica bolting is not included in data statistics.

TABLE 6 incidence of root rot and pockmark disease in Angelica sinensis treated differently

Incidence of disease	Experimental group	Control group
			Root rot (%)	0*	30±2.3
Mottle disease (%)	5±0.8*	20±1.6

The occurrence of root rot and pockmark disease of angelica treated differently is shown in table 6, and the incidence rate of root rot and pockmark disease of angelica when the bacterial liquid of lecanicillium kurosou BMDBLJJ-1 is poured in the experimental group is significantly lower than that of root rot and pockmark disease of angelica when the bacterial liquid of lecanicillium kurosou BMDBLJJ-1 is not poured in the control group. Therefore, the lecanicillium kuwana BMDBLJJ-1 bacterial liquid can be used for preventing and treating the root rot and the numb mouth disease of the angelica.

In conclusion, the lecanicillium kurosou BMDBLJJ-1 claimed by the invention can obviously reduce the incidence rate of the root rot of fritillaria, improve the green turning rate and obviously reduce the incidence rate of the root rot and the numb mouth disease of angelica, and can be used for preventing and treating the root rot and the numb mouth disease of medicinal plants.

Sequence listing

<110> institute of biological research of science institute of Gansu province

<120> Ericerus pela capable of inhibiting fusarium pathogenic bacteria and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 575

<212> DNA

<213> Ericerus pela (Lecanicilliumpsalliotae)

<400> 1

gaagagttca ctccaaccct tatgtgacat accataatgt tgcttcggcg gactcgcccc 60

ggcgtccgga cggcctagcg ccgcccgcgg cccggaccca ggcggccgcc ggagaccacc 120

aaaactattt tgtatcagca gttttttctg aatccgccgc aaggcaaaac aaatgaatca 180

aaactttcaa caacggatct cttggttctg gcatcgatga agaacgcagc gaaatgcgat 240

aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac attgcgcccg 300

ccagcattct ggcgggcatg cctgttcgag cgtcatttca accctcgact tccctttggg 360

gaaatcggcg ttggggactg gcagcatacc gccggccccg aaatggagtg gcggcccgtc 420

cgcggcgacc tctgcgtagt aatccaacct cgcaccggaa ccccgacgtg gccacgccgt 480

aaaacacccc actttctgaa cgttgacctc ggatcaggta ggaatacccg ctgaacttaa 540

gcatatcaat aaagcggagg aaagccttga tccca 575

Claims

1. A lecanicillium kuwanense (Lecanicilliumpsilotae) BMDBLJJ-1 strain is preserved in the China general microbiological culture collection management center in 2019, 10 and 11 days, and the preservation number is CGMCC No. 18585.

2. The lecanicillium mortierum BMDBLJJ-1 of claim 1, wherein the lecanicillium mortierum BMDBLJJ-1 has the amino acid sequence set forth in SEQ ID NO:1, or a sequence of ITS.

3. The use of lecanicillium mortierum BMDBLJJ-1 according to claim 1 or 2 for inhibiting fusarium oxysporum or fusarium solani.

4. The use of lecanicillium kuwanense BMDBLJJ-1 as claimed in claim 1 or 2 for preventing and treating diseases of fritillaria and/or angelica caused by fusarium, wherein the diseases are one or more of rot, blight and numb mouth disease caused by fusarium.

5. The use of claim 4, wherein the disease is fritillary root rot, angelica root rot or angelica nummularia.

6. A pesticide for controlling plant diseases caused by fusarium pathogenic bacteria, which is characterized in that the pesticide contains the lecanicillium kuwana BMDBLJJ-1 of claim 1 or 2.

7. The pesticide of claim 6, wherein said pesticide contains a spore number of 10⁶～10⁸Lecanicillium lecanii BMDBLJJ-1/ml.