CN107699526B

CN107699526B - Actinomycete strain for preventing and treating gray mold and application thereof

Info

Publication number: CN107699526B
Application number: CN201711157296.8A
Authority: CN
Inventors: 李培谦; 冯宝珍
Original assignee: Yuncheng University
Current assignee: Yuncheng University
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2021-05-25
Anticipated expiration: 2037-11-20
Also published as: CN107699526A

Abstract

The invention discloses an actinomycete strain for preventing and treating gray mold and application thereof. The strain is named as Streptomyces clavuligerus (Streptomyces junghhensis) LA-5, is preserved in China Center for Type Culture Collection (CCTCC) in 2017 at 11 and 13 months, and has the preservation number of CCTCC NO: m2017683. The fermentation product of the actinomycete can inhibit various plant pathogenic fungi and bacteria, has good inhibition effect on botrytis cinerea, cucumber fusarium wilt, cotton fusarium wilt, pythium aphanidermatum, rhizoctonia solani and other fungi, and also has certain biological activity on cauliflower soft rot bacteria, tomato ralstonia solani and other pathogenic bacteria. The strain has simple culture conditions, is easy for industrial production, can be used for developing novel efficient, broad-spectrum, low-toxicity and environment-friendly biopesticide, and has good development and application prospects.

Description

Actinomycete strain for preventing and treating gray mold and application thereof

Technical Field

The invention belongs to the technical field of crop disease control, and particularly relates to an actinomycete strain for controlling gray mold and application thereof.

Background

Crop gray mold is a worldwide fungal disease caused by Botrytis cinerea (Botrytis cinerea) and is common throughout the country. The host range of pathogenic bacteria is very wide, and the pathogenic bacteria can infect more than 200 crops including tomatoes, cucumbers, hot peppers, strawberries, grapes and the like, and not only are serious in the growth period of the plants, but also are serious in the storage and transportation processes after picking. At present, the resistance of botrytis cinerea to traditional bactericides is one of the most troublesome problems in agricultural production. Secondly, in recent years, the use of a large amount of chemical pesticides leads to the problems of limited export of agricultural products, drug resistance, environmental pollution and the like, and also poses serious threats to ecological environment, human health and the like. At present, the development of novel high-efficiency, broad-spectrum, low-toxicity and environment-friendly bactericides is always a difficult point and a hotspot for preventing and controlling crop diseases. Therefore, the development of a biogenic fungicide alternative to chemical fungicides has been delayed in the control of Botrytis cinerea.

Actinomycetes (Actinomycetes) are microorganisms with important economic value and practical value, are abundant in variety and widely distributed in nature. The soil antagonistic actinomycetes has the advantages of environmental friendliness, lasting effect, strong pertinence and the like, and shows good application prospect. Therefore, the actinomycetes with antagonistic action on the botrytis cinerea is separated from the tomato rhizosphere soil, has important significance for preventing and treating the disease, and meets the sustainable development requirement of agriculture.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an actinomycete strain, and the actinomycete fermentation liquor can inhibit various plant pathogenic fungi and bacteria and can be used for developing novel efficient, broad-spectrum, low-toxicity and environment-friendly biopesticide.

It is still another object of the present invention to provide a method for preparing the fermentation product of actinomycetes.

It is still another object of the present invention to provide a fermentation product obtained by the fermentation method of said actinomycetes.

Another object of the present invention is to provide the use of said actinomycetes and/or fermentation products thereof for inhibiting pathogenic bacteria.

The purpose of the invention is realized by the following technical scheme:

an actinomycete, named Streptomyces junghhensis (Streptomyces junghhensis) LA-5, with the preservation number of CCTCC NO: m2017683, deposited at the China center for type culture Collection at Wuhan university, Wuhan, China on 11/13/2017.

The strain is an actinomycete separated from soil collected by a vegetable greenhouse in PingLu county of Shanxi fortune.

The morphological characteristics of the actinomycete are as follows: on a Gao's medium I, the growth is good, the bacterial colony is flat and black brown after being cultured for 10 days, the color of spore silk is black gray, coffee soluble pigment is produced, and the back of the bacterial colony is dark brown; observed under a scanning electron microscope, the spores are in a short cylindrical shape, and have smooth surfaces and are chain-grown; the axial length is about 0.6 μm and the radial length is about 1.0. mu.m.

The physiological and biochemical characteristics of the actinomycetes are as follows: the strain can liquefy gelatin, hydrolyze starch, and grow on cellulose to generate hydrogen sulfide; rhamnose, mannose, sucrose, D-fructose, D-xylose and D-glucose can be used; inositol, arabinose, sorbitol, raffinose and mannitol cannot be utilized.

The 16S rDNA gene characteristics of the actinomycetes are as follows: the 16S rDNA sequence is shown in the nucleotide sequence of SEQ ID No.1, and the sequence length is 1391 bp.

The strain is classified into Streptomyces chrysotium (Streptomyces junghensis) through morphological characteristics, culture characteristic observation and molecular biology (the strain is cultured, then DNA is extracted, amplified and the sequence of 16S rDNA is determined, phylogenetic tree analysis is constructed) research and identification.

The preparation method of the actinomycete fermentation product comprises the following steps:

(1) preparing a seed solution: inoculating the activated actinomycetes to a fermentation culture medium, and culturing at the temperature of 28 ℃ and at the rpm of 160-180 for 48-60 hours to obtain a seed solution;

(2) fermentation culture: inoculating the strain into a fermentation medium with the inoculation amount of 10% (v/v), bottling with the inoculation amount of 60mL/250mL, and performing shake culture at 28 ℃ and 180r/min for 7 days to obtain the actinomycete fermentation product.

The fermentation medium is as follows: 20g of soluble starch, 1g of yeast powder, 0.5g of NaCl and K₂HPO₄ 0.5g，MgSO₄0.5g，FeSO₄0.01g, 1000mL of water, and pH of 7.2-7.4.

The activating mode is preferably as follows: culturing in the Gao's first culture medium at 28-30 deg.c until the hypha and spore grow mature.

The formula of the Gao's first culture medium is preferably as follows: KNO₃ 1g，K₂HPO₄ 0.5g，MgSO₄ 0.5g，NaCl 0.5g，FeSO₄0.01g, 20g of soluble starch, 15g of agar powder and 1000mL of distilled water, wherein the pH value is 7.2-7.4; sterilizing at 115-121 ℃ for 30 min.

The fermentation product prepared by the preparation method.

The actinomycete and/or the fermentation product thereof has biological activity of inhibiting various plant pathogenic bacteria.

The plant pathogenic bacteria comprise plant pathogenic fungi and/or plant pathogenic bacteria.

The crop pathogenic bacteria include at least one of Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum f.sp.cuminerinum), Pythium aphanidermatum (Pythium aphanidermatum), Phytophthora capsici (Phytophthora capsici), cotton Fusarium oxysporum f.sp.vassitinfectium), apple anthracnose (Colletotrichum gloeosporioides), corn leaf spot pathogen (mycosphaerotheca maydis), Rhizoctonia solani (Rhizoctonia solani), tomato Pseudomonas solani (pseudomonads solani), cauliflower soft rot pathogen (Rhizoctonia carotovora subsp.carina), potato Phytophthora nigra (Rhizoctonia carotovora subsp.sp.sp.carinica), and Phytophthora parasitica (Phytophthora rosea Phytophthora sp.c.var.sp.sp.sp.sp.var.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.a).

The actinomycete and/or a fermentation product thereof are applied to the preparation of the plant disease microorganism bactericide.

A microbial bactericide for preventing and treating plant diseases contains the said actinomycetes and/or its fermentation product.

The application method of the microbial bactericide is preferably spraying.

The plant disease is preferably gray mold.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a novel actinomycete strain LA-5. The strain has wide antibacterial spectrum and certain inhibition effect on various plant pathogenic fungi and bacteria. The strain fermentation liquor not only has good inhibition effect on botrytis cinerea, fusarium oxysporum f.sp.cubense, rhizoctonia solani and other fungi, but also has certain biological activity on cauliflower soft rot bacteria, tomato ralstonia solani and other pathogenic bacteria. The strain can be used as a biological material for developing a microbial bactericide with high efficiency, broad spectrum, low toxicity and environmental protection.

(2) The invention also provides a preparation condition of the fermentation liquor with the bactericidal activity of the strain LA-5. Fermentation conditions are as follows: culturing at 28 deg.C and 180r/min for 7 d; the culture medium comprises the following components: 20g of soluble starch, 1g of yeast powder, 0.5g of NaCl and K₂HPO₄ 0.5g，MgSO₄ 0.5g，FeSO₄0.01g, 1000mL of water, and pH of 7.2-7.4.

(3) The biocontrol actinomycete LA-5 is separated from rhizosphere soil of tomatoes in a vegetable greenhouse, is harmoniously compatible with soil ecology, and is beneficial to fully exerting the advantages of strains. The biocontrol actinomycete strain has simple culture condition, is easy for industrial production and has good development and application prospect.

Drawings

FIG. 1 is a colony morphology of actinomycete strain LA-5 according to the present invention.

FIG. 2 is a photograph of LA-5 spores of actinomycete strain according to the present invention; wherein A is the spore morphology under an optical microscope, and B is the spore morphology under a scanning electron microscope.

FIG. 3 is a graph showing the bacteriostatic effect of actinomycete strain LA-5 on Botrytis lycopersicum plate.

FIG. 4 is a graph showing the inhibitory effect of the fermentation broth of actinomycete strain LA-5 on Botrytis lycopersici.

FIG. 5 is a phylogenetic tree diagram of actinomycete strain LA-5 and related strains according to the 16S rDNA sequence.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The media formulations used in the examples were as follows:

gao's first medium: KNO₃ 1g，K₂HPO₄ 0.5g，MgSO₄ 0.5g，NaCl 0.5g，FeSO₄0.01g, 20g of soluble starch, 15g of agar powder and 1000mL of distilled water, wherein the pH value is 7.2-7.4;

a Chao's medium: sucrose 30g, NaNO₃ 2g、K₂HPO₄ 1g、FeSO₄0.01g, agar 15g, MgSO₄·7H₂0.5g of O, 0.5g of KCl, 1000mL of distilled water and pH of 7.2-7.4;

glucose aspartyl agar medium: glucose 10g, agar 15g, K₂HPO₄0.5g, 0.5g of asparagines and 1000mL of distilled water, wherein the pH value is 7.2-7.4;

starch ammonium agar medium: (NH)₄)₂SO₄ 2g、CaCO₃ 3g、K₂HPO₄1g, 10g of starch and MgSO₄1g of agar, 15g of agar, 1g of NaCL and 1000mL of distilled water;

glucose yeast extract medium: 10g of glucose, 10g of yeast extract, 4g of beef extract, 4g of peptone, 2.5g of NaCL and 1000mL of distilled water;

PDA culture medium: soaking potato juice (200 g of potato is cut into small pieces and is put into 1000mL of water to be boiled for 1 hour, then filtering is carried out to obtain filtrate, and the filtrate is diluted with water to reach the total volume of 1000mL), 15g of agar, 10g of glucose and the pH value of 7.2-7.4;

oat agar medium: oat flour 20g, saline solution 1mL (MgSO)₄·7H₂O 0.1g、MnCl₂·4H₂O 0.1g、ZnSO₄·7H₂0.1g of O, 1000mL of distilled water), 18 percent of agar, and supplementing the agar with the distilled water to 1000 mL;

NA medium: 3g of beef extract, 1g of yeast extract, 5g of peptone, 10g of glucose and 15g of agar, wherein the volume is constant to 1000mL and the pH value is 7.2-7.4.

EXAMPLE 1 isolation and characterization of Strain LA-5

1. Collection of soil samples

Collecting rhizosphere soil in a depth of 5-10 cm of a tomato plot from a certain vegetable planting greenhouse in PingLu county of the transport city of Shanxi province, subpackaging and marking, bringing back to a laboratory, and naturally drying for later use.

2. Isolation of Actinomycetes

The separation was performed by plate dilution. Grinding the air-dried soil sample by using a mortar, weighing 10g of the ground air-dried soil sample, pouring the weighed ground air-dried soil sample into a triangular flask filled with 90mL of sterile water, shaking the ground air-dried soil sample for 30min at the temperature of 28 ℃ at 180r/min, and standing the ground air-dried soil sample for 5 min to obtain a stock solution. Taking 1mL of stock solution, diluting with 10-fold gradient, and preparing into 10^-2、10^-3、10^-4、10^-5、10^-60.2mL of each suspension was added to the culture medium of Gao's No. one (containing 30mg/L of K)₂GrO₇) And uniformly coating the mixture on a flat plate, culturing and observing at 28 ℃, and picking different single colonies after 5-7 days for streaking and purifying. Transferring the purified strain to a Gao's first slant culture medium for culture, storing at 4 ℃ for later use, and co-separating to obtain 15 actinomycetes. The plate antagonist of example 3 was usedAnd (4) performing an anti-bacteria zone experiment and a bacteriostatic zone experiment, and screening to obtain the actinomycete LA-5 with a wide antibacterial spectrum.

3. Identification of Strain LA-5

(1) Morphological feature observation

The strain LA-5 grows well on most culture media (see table 1), a colony on a Gao's No. I culture medium is flat and black brown, the color of spore silk is black gray, coffee soluble pigment is produced after the strain is cultured for 10 days (figure 1), and the back of the colony is dark brown; the base silk is not broken under an optical microscope, the aerial hyphae form spore silk, and the spore silk is straight and flexible; observed under a scanning electron microscope, the spores are in a short cylindrical shape, and have smooth surfaces and are chain-grown; the axial length was about 0.6 μm and the radial length was about 1.0. mu.m (FIG. 2).

TABLE 1 culture characteristics of LA-5 strains

Note that +++ indicates good growth, + indicates good growth, and + indicates good growth

(2) Physiological and biochemical characteristics

The characteristics of strain such as gelatin liquefaction, starch hydrolysis, coagulation and peptonization of milk, cellulose hydrolysis, carbon source utilization, etc. were determined by reference to the method of Lechevalie (Lechevalier et al, 1980).

The strain can liquefy gelatin, hydrolyze starch, and grow on cellulose to generate hydrogen sulfide; rhamnose, mannose, sucrose, D-fructose, D-xylose and D-glucose can be used; inositol, arabinose, sorbitol, raffinose and mannitol cannot be utilized.

(3) Molecular identification of strain LA-5 by 16S rDNA gene sequencing method

The 16S rDNA sequencing was performed by Shanghai Bioengineering Co., Ltd.

The specific sequencing method is as follows: extracting genome DNA from fresh thallus by lysozyme method, amplifying 16S rDNA by using universal primer (27F and 1492R), detecting and sequencing PCR product, wherein the 16S rDNA sequence is as follows:

GCTCCTCCCCACAAGGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACAGGCTTTTTGAGATTCGCTCCGCCTCGCGGCTTCCCAGCTCATTGTACCTGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTCCCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACCCGACCACAAGGGGGGCCGTATCTCTACGGCTTTCCGGGCGATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCACACTCTAGCCTGCCCGTATCGACTGCAGACCCGGGGTTAAGCCCCGGGCTTTCACAACCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCTTCTACCTCACCAACTAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCGGAGCTTTCAACCTTCTCCCATGCGAGAGAAAGTGTCATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCCGAAGGGCTTCATCGTTCGACTGCA

the total length of the 16S rDNA nucleotide complete sequence of the strain LA-5 is 1391 bp. The obtained sequences are submitted to a GenBank database for BLAST analysis and comparison, strains with higher homology with LA-5 belong to the genus Streptomyces, and 16Sr DNA sequences of 12 typical strains are selected to construct a phylogenetic tree by MEGA 6.0 software. The results show that the strain LA-5 belongs to the same branch as Streptomyces junghhensis (FIG. 5).

The strain LA-5 is named as Streptomyces junghhensis (Streptomyces junghhensis) LA-5, and is preserved in the China type biological preservation center at Wuhan university in Wuhan, China at 11 and 13 months in 2017, with the preservation number of CCTCC NO: m2017683.

EXAMPLE 2 fermentation of LA-5 Strain

(1) Inoculating the strain to a Gao's first slant culture medium, and culturing at 28-30 ℃ until hypha and spores grow mature;

(2) inoculating hypha and spores on the inclined plane into a fermentation culture medium, and culturing at the temperature of 28 ℃ and the rpm of 160-180 for 48-60 h to obtain an actinomycete LA-5 seed solution;

the fermentation medium comprises the following components: 20g of soluble starch, 1g of yeast powder, 0.5g of NaCl and K₂HPO₄0.5g，MgSO₄ 0.5g，FeSO₄0.01g, 1000mL of water, and pH of 7.2-7.4.

(3) Fermentation of actinomycetes LA-5: inoculating 10% (v/v), bottling 60mL/250mL, culturing at 28 deg.C and 180r/min for 7 days to obtain 10% (v/v) concentrate⁸And (3) fermenting the LA-5 strain fermentation liquor with CFU/mL, and fermenting the culture medium in the same step (2).

EXAMPLE 3 determination of the bacteriostatic Activity of Strain LA-5

The antagonistic determination of the strain LA-5 on pathogenic fungi is carried out by adopting a plate confronting culture method, firstly 7mm fungus cakes of the pathogenic fungi to be detected are inoculated in the center of a PDA culture medium plate, 2cm around the colonies are evenly inoculated with actinomycete cakes of the strain actinomycete, the bacteriostatic effect is checked after the strain is cultured for 3 days at 26 ℃, the experimental result shows that the strain LA-5 on botrytis cinerea (the strain is disclosed in the literature of Wangmei, Heyuchun, Liuhuiping, and the like. the disease prevention mechanism of the endophytic bacillus circulans Jcx 8 on the botrytis cinerea is researched [ J ]. China ecological agriculture report, 2010,18(1): 98-101.), cucumber Fusarium wilt (the strain is disclosed in the literature of Focl Ve1 inus ecological disease promoter, flamentulous growth, bioofilm formation, and Fusarium oxysporum f.m.f.mer actinomycete.Sciensis, melon and melon of the university of the family of Japan scientific research, and the scientific research result is screening the Fusarium oxysporum melons strains (the scientific research of the university of the northern melon and fruit Science) of the Japan scientific research of the melon and fruit Academic edition), 2009,37(05):144-, 2017,25(02): 130-; taking Botrytis cinerea as an example, the bacteriostatic effect is shown in FIG. 3.

And then, determining the inhibition effect of the LA-5 strain fermentation liquor on the botrytis cinerea by adopting a paper filter strip method. Fermenting 50mL of the fermented solution for 7 days and 10 days⁸The fermentation liquid of the LA-5 strain (prepared in example 2) with CFU/mL is put into a 50mL centrifuge tube, centrifuged at 8000r/min to obtain the supernatant, and then the supernatant is filtered and sterilized by a microporous filter membrane (0.22 μm) to obtain sterile fermentation liquid, and the sterile fermentation liquid is stored in a sterilization container at 4 ℃ for later use. And infiltrating the sterile filter paper strips with fermentation liquor, symmetrically sticking the sterile filter paper strips in a PDA culture medium after fully drying, inoculating a tomato botrytis cinerea cake at the center of a culture dish, and taking sterile water as a reference. After 3 days, the colonies in the experimental group were long and narrow, the growth was hindered, and the control group had no significant effect (FIG. 4).

Activity assay for pathogenic bacteria: the test strains include bacterial wilt of tomato (which has been disclosed in the document "screening of endophytic antagonistic bacteria against tomato bacterial wilt. Phytopathology report, 2003, (04): 364. 367."), cauliflower soft rot bacterium (which has been disclosed in the document "screening of migratory mutants of Erwinia carotovora subsp. carotovora. Phytopathology report, 2000, (02): 176. 180."), potato black shank bacterium (which has been disclosed in the document "identification of pathogen of Kangchow potato black shank. Phytopathology report, 2015,45(05): 449. 454."), bacterial gummosis bacterium (Pseudomonas syringae pv. syringae, which has been disclosed in the document "Xuxylo, Emamocarb, Chengnin, et al.,. molecular identification and pathogenicity detection of sweet cherry gummosis bacterium [ J ]. Phytopathology report, 45(4): 350. 355.", i.e. corresponding to strain p.syringae pv.syringae) in this document. 1mL of pathogenic bacteria suspension is sucked and added into the NA culture medium with the temperature of about 50 ℃, the mixture is shaken evenly, and the mixture is quickly poured into a culture dish until the culture medium is solidified. Sterilizing a filter paper sheet with the diameter of about 6mm, dipping in sterile hair liquid, airing and pasting on a culture medium, pasting 5 filter paper sheets in each dish, repeating for 3 times, and taking the filter paper sheet without any filtrate as a blank control. Culturing at 30 ℃, culturing for 1-2 days, observing the inhibition zone with clear edge and recording the size, and the experimental result shows that the LA-5 fermentation broth has certain inhibition activity on pathogenic bacteria to be tested (Table 2).

TABLE 2 bacteriostatic effect of Actinomycetes LA-5 fermentation broth

Example 4 biological Activity of Strain LA-5 and its applications

1. A hypha growth rate method (Murui, 1994) is adopted, pathogenic fungus strains to be tested are cultured on a culture medium plate for 5-7 days in a 26 ℃ incubator, and then fungus cakes with the diameter of 7mm are punched at the edges of bacterial colonies by a puncher. Adding the sterile fermentation liquor into a PDA culture medium at 50 ℃ according to the proportion of 1:19, uniformly mixing and pouring into a culture dish. After cooling, the pathogenic fungi cake is inoculated, and the pathogenic fungi to be tested growing on the PDA flat plate without adding the fermentation liquid are used as a reference. Each treatment was repeated 4 times, cultured at 26 ℃ for 5 days, and when colonies of the control group grew over the plate, the colony diameter was measured by the cross method, and the average hyphal growth inhibition ratio of the strain was calculated according to the formulas A and B, and the results are shown in Table 3.

(A) Diameter of bacterial colony (mm) — measured diameter-diameter of bacterial cake

(B) Hypha growth inhibition rate ═ [ (control colony diameter-treated colony diameter)/control colony diameter ] × 100%

TABLE 3 bacteriostatic effect of LA-5 strain fermentation liquid on 8 kinds of pathogenic fungi

2. Prevention and treatment effect of strain LA-5 fermentation liquor on tomato gray mold

The prevention and treatment effect of LA-5 fermentation broth on tomato gray mold is determined by a tissue method.

The first set of experiments: 30 tomatoes which are about 5cm in diameter, similar in size and healthy in growth are selected and evenly divided into 3 groups, and the tomatoes are sprayed with LA-5 strain sterile fermentation liquor, 1500-fold liquid of 2% (effective component) propamidine aqueous solution and 4000-fold liquid of 50% (effective component) fludioxonil wettable powder serving as protective agents respectively, wherein the spraying amount is just uniform and no drop is generated. After drying in the air 10⁶The tomato botrytis cinerea spore suspension is inoculated to the minimally invasive wounds of fruits, and the inoculation amount is preferably that the spraying is uniform and no dripping is generated.

The second set of experiments: the order of inoculation and spraying is reversed, inoculation 10 being preceded by⁶And spraying the fermentation liquor and the pesticide with the same concentration as the treatment group after 24 hours per mL of the pathogenic bacteria spore suspension, setting three times for two experiments by taking sterile water as a Control (CK), measuring and recording the area of the scab after 5 days, and calculating the control effect. The results are shown in Table 4, which shows that the fermentation liquor of the strain LA-5 has better control effect on tomato gray mold.

TABLE 4 protective and therapeutic control of Botrytis cinerea with LA-5 fermentation broth

According to the results of the embodiment, the fermentation liquor of the strain LA-5 is cultured by liquid fermentation, the fermentation liquor contains substances for inhibiting common crop pathogenic bacteria, the inhibition rate of the strain LA-5 on botrytis cinerea and part of soil-borne fungi (such as cucumber fusarium wilt bacteria, melon and fruit pythium aphanidermatum, rhizoctonia solani and cucumber fusarium wilt bacteria) hyphae exceeds 80%, the occurrence of diseases can be obviously reduced, and the strain LA-5 is expected to be developed into a novel microbial agent for preventing and treating crop diseases.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> college of fortune city

<120> actinomycete strain for preventing and treating gray mold and application thereof

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cgtcagtaat ggcccagaga tccgccttcg ccaccggtgt tcctcctgat atctgcgcat 780

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tgcagacccg gggttaagcc ccgggctttc acaaccgacg tgacaagccg cctacgagct 900

ctttacgccc aataattccg gacaacgctt gcgccctacg tattaccgcg gctgctggca 960

cgtagttagc cggcgcttct tctgcaggta ccgtcacttg cgcttcttcc ctgctgaaag 1020

aggtttacaa cccgaaggcc gtcatccctc acgcggcgtc gctgcatcag gctttcgccc 1080

attgtgcaat attccccact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140

tgtggccggt cgccctctca ggccggctac ccgtcgtcgc cttggtgagc ttctacctca 1200

ccaactagct gataggccgc gggctcatcc tgcaccgccg gagctttcaa ccttctccca 1260

tgcgagagaa agtgtcatcc ggtattagac cccgtttcca gggcttgtcc cagagtgcag 1320

ggcagattgc ccacgtgtta ctcacccgtt cgccactaat ccaccccgaa gggcttcatc 1380

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Claims

1. An actinomycete, which is characterized in that: is named as Streptomyces glaucescens (Streptomyces junghhensis) LA-5, and the preservation number is CCTCC NO: m2017683, deposited at the China center for type culture Collection at Wuhan university, Wuhan, China on 11/13/2017;

the 16S rDNA sequence of the Streptomyces glaucescens (Streptomyces chunghensis) LA-5 is shown as SEQ ID NO: 1 is shown.

2. The method for producing a fermentation product of actinomycetes according to claim 1, comprising the steps of:

3. The method for producing a fermentation product of actinomycetes according to claim 2, wherein the fermentation medium is:

20g of soluble starch, 1g of yeast powder, 0.5g of NaCl and K₂HPO₄0.5g，MgSO₄0.5g，FeSO₄0.01g, 1000mL of water, and pH of 7.2-7.4.

4. The method for producing a fermentation product of actinomycetes according to claim 2, wherein said activation is carried out by:

culturing the actinomycetes in a Gao's first culture medium at the temperature of 28-30 ℃ until hypha and spores grow mature.

5. A fermentation product of actinomycetes, characterized in that:

the fermentation product of the actinomycetes according to any one of claims 2 to 4.

6. Use of the actinomycetes and/or the fermentation product thereof according to claim 1, wherein:

the application is the application in the preparation of plant disease microbial bactericides;

the plant diseases are plant diseases caused by plant pathogenic bacteria, and the plant pathogenic bacteria are one or more of botrytis cinerea, fusarium oxysporum of cucumber, pythium aphanidermatum, phytophthora capsici, fusarium wilt of cotton, corn leaf spot pathogen, apple anthracnose pathogen, rhizoctonia solani, tomato ralstonia solani, cauliflower soft rot pathogen, potato phytophthora parasitica and bacterial gummosis.

7. A microbial fungicide for controlling plant diseases is characterized in that:

the microbial fungicidal agent for controlling plant diseases contains the actinomycetes described in claim 1 and/or the actinomycetes fermentation product described in claim 2.

8. The microbial fungicide for controlling plant diseases according to claim 7, characterized in that:

the application method of the microbial bactericide is spraying.