CN113684150A - An Endophytic Actinomycetes HJG-5 of Zanthoxylum and Its Application - Google Patents

Abstract

The invention discloses a pepper endophytic actinomycete HJG-5 and application thereof in producing growth promoting active substances and broad-spectrum antibacterial active substances. The invention has the advantages that: the pepper endophytic actinomycetes HJG-5 is a strain which is separated, purified and screened from various tissues of pepper and has wide antibacterial spectrum and obvious antibacterial effect; the fermentation liquor of the strain HJG-5 contains growth promoting active substances and broad-spectrum antibacterial active substances, and lays a foundation for the development and research of rich natural resources of biological pesticides and microbial pesticides.

Description

Prickly ash endophytic actinomycete HJG-5 and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation and application, and particularly relates to a pepper endophytic actinomycete HJG-5 and application thereof.

Background

Plant diseases are one of main factors threatening agricultural production, and in the agricultural production of China, chemical synthetic pesticides always occupy a main position in disease control due to the characteristics of wide antibacterial spectrum, high control efficiency and quick response, but the 3R problem caused by long-term overuse, abuse and misuse is more and more serious. With the deep mind of the concept of sustainable development and the concern of people on food safety, a new prevention and control approach which is safe, efficient and accords with the concept of sustainable development is sought, and the problem to be solved for preventing and controlling plant diseases is provided. The microorganism or metabolite thereof is utilized to prevent and treat diseases, and the microorganism or metabolite thereof has the characteristics of high efficiency, low toxicity, strong selectivity, small influence on the ecological environment, increase in both production and income and the like, and becomes a current research hotspot. The endophytic actinomycetes and the host form a harmonious relation in the symbiotic process, the host is wide, the variety is complex and various, the generated bacteriostatic active substance is various in variety, and the application prospect is wide. Verma et al isolated three strains of endophytic streptomycete from root tissue of Azadirachta indica A. Juss, found that the suspension treated seeds can significantly promote the growth of the plants and inhibit the growth and reproduction of Alternaria alternata pathogens. Misk et al isolated 11 strains of endophytic actinomycetes from various plants in Australia, found that they all inhibited the growth of Phytophthora root rot, and strain WRA1 significantly promoted plant growth. The King Zhenzhen and the like divide and screen out an endophytic actinomycete streptomyces mairei OsiRt-1 which can obviously inhibit the growth of rice blast germs from rice plants, and the control effects on rice seedling blast and panicle blast under field conditions are respectively 7.76 percent and 25.65 percent. Therefore, screening the plant endophytic antagonistic actinomycetes, researching the biocontrol effect and the biocontrol mechanism of the plant endophytic antagonistic actinomycetes and having important practical significance for the practical production and application of microbial pesticides.

The pricklyash peel can be used as raw material of spice and also can be used as medicine. The medicinal value is recorded in Shennong Ben Cao Jing and Ben Cao gang mu, and can dispel cold and dampness, warm middle energizer and alleviate pain. The Hansheng man finds that the pricklyash peel volatile oil has stronger inhibition effect on HeLa, A549 and K562. One of the main components of the compounds extracted from pepper such as Ge, PauL and the like can inhibit the oviposition of the wheat moth and has strong antifeedant effect on the larva of the wheat moth. Singh et al found that zanthoxylum bungeanum essential oil cannot control the propagation of rice weevils but can protect wheat from being damaged. Navarete and the like find that the quantity of intestinal nematodes in sheep can be reduced after pepper stem bark is decocted, and the chloroform extract alpha-sanshool has severe toxicity to roundworms. Paik et al found that volatile substances in the peel of zanthoxylum bungeanum can inhibit HepG2 liver cancer cells. However, no relevant report is found in the current research on the actinomyces capsici.

In recent years, the problems of limited export of agricultural products, drug resistance, environmental pollution and the like are caused by the large amount of chemical pesticides, and the development of novel efficient, broad-spectrum, low-toxicity and environment-friendly biological bacteriostatic agents is urgently needed.

Disclosure of Invention

The invention provides a pepper endophytic actinomycete HJG-5, wherein the fermentation liquor of the strain HJG-5 contains a growth promoting active substance which can promote the growth of plants, and the fermentation liquor contains a high-efficiency, broad-spectrum, low-toxicity and environment-friendly bacteriostatic active substance which can play a good bacteriostatic role.

A pepper endophytic actinomycete HJG-5 is named as: HJG-5, class name: streptomyces cinnamomi streptomyces cinnanocastanaceus with a preservation number of: CGMCC No.14969, preservation date: and 11, and 30 days in 2017, wherein the preservation unit is as follows: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing.

Further, it was confirmed through molecular biological studies that the HJG-5 strain can be classified as S.cinnamomi.

Furthermore, the zanthoxylum bungeanum endophytic actinomycete HJG-5 strain is cultured on an oat flour agar culture medium for 2-3 days, the surface of a bacterial colony is smooth and white, white aerial hyphae grow from the edge of the bacterial colony after the culture is carried out for 6 days, and the bacterial colony gradually changes into light gray after 8 days; the colony morphology is conical uplift in the culture process, the edge of the colony is provided with 4-6 circles of concentric ring veins, and the periphery is provided with a halo-shaped soil edge; microscopic observation shows that aerial hyphae are developed and have more branches, and hyphae in the substrate have no transverse septa and are not broken.

Furthermore, the culture characteristics of the zanthoxylum bungeanum endophytic actinomycete HJG-5 strain are that the surfaces of colonies on ISP2, PDA and nutrient agar are raised to be compact fluffy powder, aerial hyphae are developed and are white or grey white, and substrate hyphae are brown yellow or yellow brown; the aerial hyphae on ISP5 are light gray, and the basal hyphae are pale goose yellow; the hyphae generated on ISP4, Chachi's and Gao's No. I culture medium are thin, mostly in light white gray color, and have no inner hyphae; hardly grows on ISP 3.

Furthermore, the pricklyash peel endophytic actinomycete HJG-5 strain can produce melanin, liquefy part of gelatin, reduce nitrate to positive, hydrolyze starch, coagulate and peptonize milk without producing H₂S, no growth on cellulose; starch, xylose, arabinose and fructose cannot be utilized, glucose, maltose, sucrose, lactose, mannitol and glycerol can be utilized.

Application of prickly ash endophytic actinomycetes HJG-5 in producing growth promoting active substance and broad-spectrum antibacterial active substance is provided.

Furthermore, the growth promoting active substances and the broad spectrum antibacterial active substances are derived from fermentation liquor obtained after the HJG-5 bacterial strains are fermented and cultured.

Furthermore, the carbon source in the fermentation medium for the fermentation culture of the HJG-5 strain is maltose, and the nitrogen source is tryptone; and maltose: the proportion of the tryptone is 2: 1.

furthermore, the culture conditions for the fermentation culture of the HJG-5 strain are 180 r.min ^-1, pH8, liquid loading 125mL/250mL, inoculum size 8%, culture temperature 28 ℃, culture time 9 d.

Furthermore, the fermentation liquor has better thermal stability, the bacteriostasis rate is maintained to be about 80% below 80 ℃, and the fermentation liquor is very stable in neutral and weak alkaline environments; the antibacterial activity is not changed by ultraviolet irradiation; the storage stability is good at 4 ℃, and after standing for 90 days, the inhibition rate of the A.solani can reach 85.15% of the antibacterial activity of the original fermentation liquor.

Furthermore, when the fermentation liquor is diluted by 20 times, the growth promoting effect and the bacteriostasis effect are optimal; after the tomato seeds are treated by the 20 times diluted fermentation liquor, the germination index is improved by 16.75 percent relative to CK; the height, the root length, the fresh weight and the dry weight of the tomato seedlings are respectively improved by 29.0 percent, 20.4 percent, 24.9 percent and 35.9 percent compared with the CK group, and the tomato seedlings are obviously increased.

The invention has the advantages that: the pepper endophytic actinomycetes HJG-5 is a strain which is separated, purified and screened from various tissues of pepper and has wide antibacterial spectrum and obvious antibacterial effect; the fermentation liquor of the strain HJG-5 contains growth promoting active substances and broad-spectrum antibacterial active substances, and lays a foundation for the development and research of rich natural resources of biological pesticides and microbial pesticides.

Drawings

FIG. 1 is a graph showing the inhibitory effect of actinomyces capsici HJG-5 on 12 types of pathogenic bacteria according to the present invention;

FIG. 2 is a graph showing the inhibitory effect of a fermentation filtrate of actinomyces capsici HJG-5 on 12 pathogenic bacteria according to the present invention;

FIG. 3 is a morphological feature diagram of a colony of Actinomyces Zanthoxyli endophytic HJG-5 and an aerial hypha according to the invention;

FIG. 4 is a diagram showing the result of electrophoresis of a 16SrDNA amplification product of actinomyces capsici HJG-5 according to the present invention;

FIG. 5 is a phylogenetic tree of Actinomyces Zanthoxyli HJG-5 of the present invention;

FIG. 6 is a graph showing the growth of Actinomyces endophytic HJG-5 and the production of bacteriostatic active substances according to the present invention;

FIG. 7 is a graph showing the effect of different basal media on the bacteriostatic activity of HJG-5 strain;

FIG. 8 is a graph showing the effect of different C sources on the bacteriostatic activity of the fermentation filtrate of HJG-5 strain;

FIG. 9 is a graph showing the effect of different N sources on the inhibition rate of fermentation filtrate of HJG-5 strain;

FIG. 10 is a graph of the analysis trend of the culture conditions in the orthogonal test;

fig. 11 is a graph of the inhibitory effect of HJG-5 strain fermentation broth on a.solani before and after optimization;

FIG. 12 is a graph of the effect of temperature on the bacteriostatic activity of the fermentation filtrate of strain HJG-5;

FIG. 13 is a graph showing the effect of pH on the bacteriostatic rate of the fermentation filtrate of HJG-5 strain;

FIG. 14 is a graph showing the effect of UV irradiation on the bacteriostatic activity of the fermentation filtrate of HJG-5 strain;

FIG. 15 is a graph showing the effect of storage time on the bacteriostatic activity of the fermentation filtrate of HJG-5 strain;

fig. 16HJG-5 strain fermentation filtrate influence on a. solani hyphal growth;

fig. 17 graph of the effect of different concentrations of strain fermentation filtrates on a. solani cell membrane permeability;

FIG. 18 is a graph of the effect of fermentation filtrate of strain 18HJG-5 on GI of tomato seeds;

FIG. 19 is a graph of the effect of fermentation broth of strain 19HJG-5 on the growth of tomato seedlings;

FIG. 20 is a graph showing the effect of fermentation broth of strain 20HJG-5 on the growth of tomato seedlings;

FIG. 21 is a graph of the in vitro prevention and treatment effect of the fermentation filtrate of the 21HJG-5 strain;

FIG. 22 is a diagram showing the results of the detection of bacteriostatic activity of silica gel column chromatography separation components.

Detailed Description

1. Test materials

1.1 plant samples

The root, stem, leaf and fruit of Zanthoxylum bungeanum is collected from Shanxi university of agriculture in 2015 and 7 months; tomato seed modified red general F1 was from west ann shuangfeng seeds llc.

1.2 test Medium

Culturing and preserving culture medium of actinomycete strain: PDA culture medium.

Fermentation medium: PD medium.

Gao's first medium: soluble starch 20g, MgSO₄·7H₂O 1g，KNO₃1 g，NaCl 0.5g，K₂HPO₄·3H₂O 0.5g，FeSO₄·7H₂0.01g of O, 20g of agar and 1000mL of distilled water, and the pH value is 7.4-7.6.

Soybean meal liquid medium: 10g of soybean meal, proteinPeptone 3g, glucose 10g, NaCl 2.5g, CaCO₃2g, 1000mL of distilled water, pH 7.2-7.4.

Millet liquid culture medium: 10g of millet, 10g of glucose, 2g of NaCl, 3g of peptone and CaCO₃ 2g，NH₄NO₃1g, 1000mL of distilled water, pH 7.2-7.4.

Liquid culture medium of soybean corn flour: 10g of soybean meal, 10g of corn flour, 5g of soluble starch and K₂HPO₄0.5g, 1000mL of distilled water, pH 7.2-7.4.

1.3 test for phytopathogenic fungi

Botrytis cinerea (Bortytis cinerea), tomato early blight (Alternaria solani), wheat gibberellic disease (Fusarium graminearum), cotton Rhizoctonia solani (Rhizoctonia solani), Sclerotinia sclerotiorum (lib.) de Bary), bean anthracnose (Colletotrichum lindlemuticum), pepper oxysporum (Fusarium oxysporum), watermelon Fusarium (Fusarium oxysporum f.sp.Niveum), apple rot (Valsa mali), carrot brown rot (leptospora libanotis), walnut rot (Monilinia laxa), peach brown rot (Monilinia fructicola), and all of the university of Shanxi agricultural and pharmaceutical laboratories.

1.4 test chemical reagents

CH₄O、C₆H₁₄、C₄H₈O₂、CHCl₃、CH₂Cl₂、CH₃(CH₂)₃OH、HCl、NaOH。

1.5 test instruments

An HZQ-QX full-temperature shaking incubator, a UV-754 ultraviolet visible spectrophotometer, an HD-920 super clean workbench, an ST16R refrigerated centrifuge, an ALPCL-32L high-temperature sterilization pot, a rotary evaporator, an S-234 analytical balance, an electric heating constant-temperature incubator and a DDS-11A conductivity meter.

2. Experimental methods

2.1 isolation and screening of antagonistic actinomycetes of zanthoxylum bungeanum endophytic

2.1.1 Collection of plant samples

Selecting root, stem, leaf and fruit of good-growing and healthy pepper plants for collection, preserving in a fresh-keeping bag at 4 ℃ in a refrigerator, marking with label paper, and treating as soon as possible within one week.

2.1.2 isolation and purification of endophytic Actinomycetes

Pretreatment of the plant sample: and (3) after the collected sample is washed clean under water flow, thoroughly cleaning impurities remained on the surface of the sample by using an ultrasonic cleaning instrument. Cutting the sample into 2cm small sections by using a sterile knife, soaking in 75% alcohol for 3min, washing with sterile water for 2 times, soaking in 3% NaClO for 3min, washing with sterile water for 3 times, soaking in 75% alcohol for 1min, washing with sterile water for 3 times, and air-drying on sterilized dry filter paper sheets.

And (3) separating the strains by a tissue homogenization method: grinding sterilized materials in 10mL sterile water, standing for 15min, collecting supernatant 80 μ L, and spreading on K-containing solution₂Cr₂O₇750 mu g/mL Gao's No.1 plate, 28 degrees C culture 2-3 weeks, every day observation record colony situation, once find have colony growth, pick up immediately, according to colony apparent characteristics, size, soluble pigment have or not on the Gao's medium streak culture, after many times of purification numbering and slope preservation.

And (3) surface disinfection result inspection: 80 μ L of the last rinsing water was taken and spread on a Gao's first plate, cultured under the same conditions, and used as a control for checking whether surface disinfection was complete.

2.1.3 screening of endophytic antagonistic Actinomycetes

(1) Preliminary screening

Adopting a four-point plate opposing method, taking B.cinerea and A.solani cultured for 6-7 days, making a fungus cake with a phi 5mm by using a puncher, inoculating actinomycetes to be tested at a position 2cm away from the center of a PDA plate in a crossed manner, culturing at 28 ℃, measuring the diameter of pathogenic bacteria after 7 days, calculating the bacteriostasis rate, inoculating the pathogenic bacteria only as CK, and repeating the experiment for 3 times per treatment.

(2) Double sieve

Inoculating actinomycetes to be detected on a PDA culture medium, culturing at 28 deg.C for 7d, and beating 2 actinomycetesThe fungus cake with a diameter of 5mm is inoculated into a seed culture solution (100mL/250mL) and treated at 28 ℃ and 160 r.min^-1Shake-flask culturing for 48h to obtain seed solution, inoculating 10% (V/V) of the seed solution into 200mL/500mL fermentation medium, and culturing at 28 deg.C for 160 r.min^-1And after shake flask culture for 7d, obtaining the fermentation liquor of the strain. 12000r min of fermentation liquor^-1Centrifuging, and filtering with 0.22 μm microporous membrane to obtain fermentation filtrate.

Adopting a growth rate method, mixing the fermentation filtrate and a PDA culture medium according to the ratio of 1: 9 mixing them uniformly to prepare a flat plate. And after the plate is solidified, inoculating an A.solani block with the diameter of 5mm into the center, culturing at 28 ℃ for 6-7 days, measuring the diameter of the bacteria by a cross method, and calculating the bacteriostasis rate. Repeat 3 times, with blank PD medium as CK.

2.2 Classification and identification of Actinomyces endophytic HJG-5

2.2.1 morphological feature Observation

Inoculating HJG-5 strain on PDA culture medium by adopting an insert method, culturing at 28 deg.C for 7d-21d, taking embedded sheets every 5d during the culture period, observing under an optical microscope, performing primary identification according to Streptomyces identification handbook and Actinomycetes classification and identification, and mainly observing colony morphology, growth morphology of intrabasal hyphae and aerial hyphae; the hypha in the substrate has the characteristics of no transverse septa, rupture and the like.

2.2.2 culture characteristic Observation

Selecting 8 culture media of Gao's 1, ISP2, ISP3, ISP4, ISP5, potato agar, nutrient agar and Italian agar, preparing a plate, inoculating HJG-5 strains, culturing at 28 ℃, and observing and recording the color and growth condition of hyphae after 5d, 7d, 10d, 15d and 21d, whether soluble pigment is generated or not and the generated color.

2.2.3 physiological and Biochemical identification

The physiological and biochemical identification of the strain HJG-5 is carried out according to the methods of Streptomyces identification handbook and Actinomycetes rapid identification and systematic classification.

2.2.4 carbon Source utilization

Based on a Pogostemon solid culture medium, carbon sources such as glucose, sucrose, lactose, maltose, xylose, arabinose, fructose, mannitol, glycerol and starch are used for preparing a flat plate, then HJG-5 strains are inoculated by streaking, and after the strain is cultured at the constant temperature of 28 ℃ for 7 days, the growth condition of the strain is observed.

2.2.5 Melanin production

The HJG-5 strain was inoculated into a tyrosine medium and incubated at 28 ℃ and observed for the presence or absence of melanin production at 7d and 14d after inoculation, respectively.

2.2.6 Hydrogen sulfide Generation

The HJG-5 strain was inoculated into a Chassis-Nam medium and cultured at 28 ℃ for 7 days, and then whether black or brown appeared was observed.

2.2.7 nitrate reduction test

Inoculating HJG-5 strain in nitrate liquid culture medium, culturing at 28 deg.C for 7-10 d, collecting one part of culture solution, adding Grignard reagent and aniline reagent, and making it become positive if it turns red or pink, and collecting another part of culture solution, adding aniline reagent, and making it become negative if it turns blue, or making it become positive if it does not turn color.

2.2.8 liquefaction of gelatin

Inoculating HJG-5 strain into gelatin culture medium, culturing at 28 deg.C for 7d-14d, and cooling in refrigerator at 4 deg.C for 30min before observation, wherein if gelatin around colony can flow, it is positive, otherwise it is negative.

2.2.9 milk coagulation or peptonization

The HJG-5 strain is inoculated into a milk liquid culture medium, cultured at 28 ℃, observed at 5d, 10d and 15d, and becomes liquid again if coagulum appears, and then the strain is positive.

2.2.10 starch hydrolysis

The HJG-5 strain is streaked and inoculated on a starch agar culture medium plate, after 7 days, iodine solution is dripped around a bacterial colony, and the situation of a transparent ring is observed, and the bacterial colony is positive if the situation exists.

2.2.11 cellulose utilization

The HJG-5 strain was inoculated on a filter paper strip soaked in the medium and cultured at 28 ℃ for 7 days, if the strain could grow on the filter paper strip, and the filter paper was decomposed and thinned to be fibrous, which was positive.

2.2.12 molecular biology identification

HJG-5 strain DNA is extracted by CTAB method, 16S rDNA gene is amplified. The primers are 27 f: 5'-AGAGTTTGATCCTG GCTCAG-3', 1492 r: 5 '-TACGGYTACCTTGTTACGACTT-3'. Amplification System and procedure the amplification products were sent to Kingzhi Biotech, Inc. for sequencing, according to the method of Coombs. After the obtained 16SrDNA sequence is BLAst on NCBI, the sequence is compared with a sequence in Genbank, a strain gene sequence with higher homology with the sequence is selected, MEGA5.1 software is used for analysis, and a Neighbor-J origin method is adopted to construct an HJG-5 strain phylogenetic tree.

2.3 fermentation culture of endophytic actinomycetes HJG-5

2.3.1 preparation of seed solutions

Activating the stored HJG-5 strain on a PDA plate, beating two phi 5mm fungus cakes, inoculating into a triangular flask with 100mL/250mL PD medium, and culturing at 28 deg.C for 160 r.min^-1And carrying out shake flask culture for 48h to obtain a seed culture solution.

2.3.2 preparation of fermentation broth

Inoculating seed solution of HJG-5 strain into PD fermentation culture medium at 28 deg.C and 160r min in an amount of 10% (V/V)^-1After 7 days of shake flask culture, obtaining the fermentation liquor of the strain, 12000 r.min^-1Centrifuging, filtering with 0.22 μm microporous membrane to obtain HJG-5 fermented filtrate, and storing at 4 deg.C. 2.3.3 detection of bacteriostatic Activity of fermentation broth

The growth rate method was used, with a.solani as indicator and CK as blank PD medium.

2.3.4 growth of the thallus and accumulation of the bacteriostatic active substances

Inoculating the seed culture solution into PD fermentation medium (250mL/500mL) at 10% (V/V), 28 deg.C, 160 r.min^-1Culturing for different time (1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d), collecting fermentation broth 100mL per day, 12000 r.min^-1Centrifuging, filtering the supernatant with 0.22 μm microporous membrane, measuring its antibacterial activity to A.solani by growth rate method, oven drying the precipitate thallus, and weighing.

2.3.5 Medium and culture Condition screening

Inoculating seed fermentation broth of HJG-5 strain to sterilized basic culture solution (A: Gaoshi No.1 liquid culture medium, B: semen Setariae liquid culture medium, C: semen glycines powder liquid culture medium, D: semen glycines semen Maydis powder liquid culture medium) at an inoculation amount of 10% (V/V)Nutrient base, E: PD medium), 28 ℃, 160 r.min^-1And 7d, measuring the bacteriostasis rate of the fermentation filtrate of the HJG-5 strain to the A.solani under different fermentation basal medium conditions, and determining the optimal fermentation basal medium.

The glucose in a basic culture medium is replaced by equivalent (20g/L) of soluble starch, maltose, sucrose, lactose, mannitol and corn flour, other components are unchanged, the fermentation basic culture medium is CK, and when different C sources are measured, the bacteriostatic activity of the fermentation filtrate of the HJG-5 strain on A.solani is determined, and the optimal carbon source is determined.

Selecting the optimum C source as C source, and adding equal amount (10g/L) of semen glycines powder, peptone, tryptone, yeast powder, beef extract, urea, (NH) respectively₄)₂SO₄、KNO₃And under different N source conditions, the bacteriostatic activity of the fermentation filtrate of the HJG-5 strain on A.solani is measured, the optimal nitrogen source is determined, and the original PD culture medium is used as CK.

To further study the effect of fermentation conditions on the production of the bacteriostatic active substance from HJG-5 strain, an orthogonal test L16 (4) was used⁵) Referring to table 1, the rotation speed, PH, liquid loading amount, inoculation amount, and fermentation time were optimized to screen out the optimum fermentation conditions for the strain HJG-5.

TABLE 1 fermentation conditions orthogonal experimental design

2.4HJG-5 Strain fermentation broth stability assay

2.4.1 measurement of thermal stability

Taking six sterile test tubes, treating each fermentation filtrate 10mL at 40 deg.C, 60 deg.C, 80 deg.C, 100 deg.C and 121 deg.C for 30min, cooling to room temperature, and measuring antibacterial activity, wherein the untreated fermentation filtrate is CK.

2.4.2 ultraviolet radiation stability determination

Respectively placing 10mL of fermentation filtrate in a sterilized culture dish, respectively irradiating for 2h, 4h, 6h, 8h, 10h, 12 h and 24h under an ultraviolet lamp (15cm), and measuring the antibacterial activity of the fermentation filtrate, wherein the fermentation filtrate without ultraviolet irradiation is CK.

2.4.3 determination of acid-base stability

10mL of the fermentation filtrate were taken in sterile tubes and the pH was adjusted to: 2.0, 3.0, 4.0, 5.0, 7.0, 8.0, 9.0, 10, 11.0 and 12.0, standing at room temperature for 2h, adjusting to original pH6.0, and measuring antibacterial activity, wherein fermentation filtrate (pH6.0) without acid-base treatment is used as control.

2.4.4 storage stability assay

Taking fermentation filtrate, storing in sterile test tube, storing in dark at 4 deg.C in refrigerator, and sampling at 0d, 15d, 30d, 45d, 60d, 75d, 90d, 105d, and 120d respectively to detect its antibacterial activity.

The detection of the antibacterial activity adopts a growth rate method, and A.solani is used as a target bacterium.

2.5 initial study on bacteriostatic action of fermentation filtrate of 2.5HJG-5 Strain

2.5.1 Effect on the growth and morphology of pathogenic hyphae

Picking the inhibited A.solani hyphae in the determination of the bacteriostatic activity on a glass slide, observing the hyphae form by microscopy, taking the normally grown A.solani hyphae as CK, recording and taking photomicrographs.

2.5.2 Effect on the cell Membrane permeability of pathogens

Inoculating cultured A.solani producing large amount of spores into PD culture solution, and culturing at 28 deg.C for 160r min^-1After 5-6 days, 7500 r.min^-1Centrifuging to collect mycelium, washing with sterile water for 3 times, weighing 5 parts, placing 5g of each part in a 100mL triangular flask, adding 50mL sterile water, adding fermentation filtrate to obtain final concentration of 5%, 10%, 13%, 20%, CK, adding equal volume of PD culture solution, and culturing at 28 deg.C for 160 r.min^-1After 5mL of the supernatant was centrifuged at 0h, 0.5h, 1h, 1.5h, 2h, 4h, 6h, 20h and 24h, respectively, the conductivity value of the supernatant was measured. The effect of the bacteriostatic active on a. solani cell membrane permeability is expressed as the change in conductivity value.

2.6 determination of growth promotion and biocontrol effects of 2.6HJG-5 strain fermentation broth

2.6.1 Effect on tomato seed Germination

Selecting tomato seeds with uniform and plump sizes, respectively soaking the tomato seeds in a stock solution of fermentation liquor, 5x, 10x, 20x, 50x, 80x and 100x diluents for 24 hours after surface disinfection, treating the tomato seeds with blank fermentation liquor to be used as CK, placing 30 granules in each dish in a culture dish paved with two layers of sterilized filter paper, carrying out dark culture at 27 ℃, uniformly adding sterile water at regular time to keep moist, repeating the treatment for 3 times, repeating the test for 2 times, and observing and measuring the germination index of each treatment within 7 days every day.

Germination Index (GI) ═ Sigma Gt/Dt

In the formula: gt: number of sprouts within t days; and Dt: the number of days of germination.

2.6.2 Effect on tomato shoot growth

Selecting tomato seeds by the method, disinfecting the surfaces of the tomato seeds, sowing the tomato seeds, selecting the seedlings with consistent growth vigor and size when fourth true leaves grow out from the seedlings, irrigating roots of the seedlings with fermentation liquor stock solution, 5X, 10X, 20X, 50X, 80X and 100X diluted fermentation liquor respectively, wherein 20mL of each plant is irrigated with the roots again after five days, culturing at room temperature by taking PD culture solution as CK, watering at proper time, and measuring plant height, fresh weight, dry weight and root length after sowing for 70 days.

2.6.3 method for determining the prevention and treatment effects of in vitro leaf

Selecting healthy leaves with similar leaf positions and consistent growth on a tomato plant, carrying out surface treatment and disinfection, then respectively spraying a fermentation liquor stock solution and 5X, 10X and 20X diluent on the leaves by adopting a needle-punching inoculation method, taking a sprayed blank PD culture solution as CK, inoculating a tomato early blight bacterium cake after 24h, placing the treated leaves in a culture dish with sterile wet filter paper paved at the bottom, repeating the test three times every six leaves, alternately culturing in a moisturizing dark place at 27 ℃, observing the disease condition of the leaves after 4d, and calculating the bacteriostasis rate.

Inhibition rate/% (control lesion area-treatment lesion area) × 100/control lesion area

2.6.4 in vitro fruit control assay

Selecting healthy tomato fruits, disinfecting with 75% alcohol, washing with sterile water for later use, scratching a wound with the diameter of 5mm in the middle of the tomato fruits by using a sterile needle by adopting a needle-stick inoculation method, respectively spraying a fermentation liquor stock solution, a 5X dilution solution, a 10X dilution solution and a 20X dilution solution, inoculating an A.solani fungus cake after 24 hours by using a sprayed blank PD as a CK, placing the inoculated A.solani fungus cake in a sterile tray with wet filter paper laid at the bottom, and wrapping and sealing the inoculated A.solani fungus cake by using a preservative film. The experiment was repeated 3 times for each 5 fruits treated, incubated at 27 ℃ and observed for fruit development after 4 days.

2.7 preliminary isolation of bacteriostatic active substances and determination of biological Activity

2.7.1 screening of optimal extraction solvent

Packing the strain HJG-5 fermentation filtrate in five triangular flasks (200mL/500mL), adding equal volume of petroleum ether, ethyl acetate, chloroform, dichloromethane, and n-butanol respectively, at 28 deg.C and 160 r.min^-1And standing for 4-6h in a separating funnel after 4h, respectively collecting an Organic phase and an aqueous phase, extracting and combining Organic phase for multiple times, performing rotary evaporation to dryness under the condition of proper temperature, dissolving with methanol and fixing the volume to 10mL, and measuring the antibacterial activity of the Organic phase to the A.solani by using a growth rate method to determine the optimal Organic extraction solvent.

2.7.2 silica gel column chromatography

2.7.2.1 preparation of crude extracts of bacteriostatic active substances

Preparing HJG-5 strain fermentation liquid 3L, and processing at 12000 r.min^-1Centrifuging, filtering with 0.22 μm microporous membrane to obtain fermented filtrate, extracting with n-butanol for three times, mixing organic phases, rotary steaming at 55 deg.C, dissolving the crude extract with 5mL of methanol, filtering with 0.45 μm organic microporous membrane, and storing in refrigerator at 4 deg.C.

2.7.2.2 silica gel column chromatography

Column assembling: the silica gel (200-mesh and 300-mesh) is mixed into paste by petroleum ether, poured into a chromatographic column (30 multiplied by 450mm), stirred while added, the column body is lightly knocked by a rubber hammer to discharge bubbles in the column, a cock is lightly screwed to slowly settle the silica gel, the silica gel is filled to 5 to 7cm away from the pipe opening, the column is sprayed by a proper amount of petroleum ether until the column surface stops descending, and meanwhile, a layer of quartz sand is uniformly covered on the surface of the column bed to protect the flatness of the surface of the column bed.

Sample adding: when the liquid surface in the column is about 1cm away from the surface of the silica gel, the prepared crude extract (2mL) is slowly and uniformly added along the inner wall of the column by using a suction pipe, and when the sample moves to the position below the surface of the silica gel, the gradient elution is sequentially carried out by using reagents with the polarity from small to large.

Elution and sample collection: the eluent is petroleum ether: chloroform (1:1), chloroform: methanol (2:1), chloroform: methanol (1:1), chloroform: methanol (1:2), methanol, 200mL per gradient eluent. The flow rate is controlled to be 3-4 drops/S, and each tube is 20 mL.

2.7.2.3 detection by silica gel thin layer chromatography

Fractions collected in the tube were checked by TLC (developing solvent chloroform: methanol 4:1, iodine sublimation as color developing reagent) and the same fractions were combined according to Rf values.

2.7.2.4 determination of biological Activity of Components

And (3) concentrating the combined components under reduced pressure, and measuring the bacteriostasis rate of substances separated from each component on the A.solani by using a bacteriostasis ring method.

3. Results and analysis

3.1 separation and screening of antagonistic actinomycetes in zanthoxylum bungeanum L and determination of bacteriostatic activity

3.1.1 isolation and purification of Actinomyces sanshoamides

And classifying and purifying the actinomycetes growing on the separation culture medium according to the apparent characteristics, the size and the existence of soluble pigments of the colonies to obtain 11 endophytic actinomycetes, wherein 8 actinomycetes are from roots, and 3 actinomycetes are from stems. No colony is found on the CK culture medium, the surface disinfection is proved to be thorough, and the obtained bacterial strains are all pepper endophytes.

3.1.2 screening of antagonistic actinomycetes in Pepper

Taking B.cinerea and A.solani as indicator bacteria, and adopting a four-point plate confrontation method to perform primary screening on 11 separated endophytic actinomycetes, wherein 6 endophytic actinomycetes have an antibacterial effect (shown in table 2), the antibacterial rate on the A.solani is 44.4-73.59%, the inhibition rate on the B.cinerea is 29.25-60.10%, and the inhibition rates of HJG-5 strains on the two kinds of pathogenic bacteria are optimal.

The 6 antagonistic bacteria strains are rescreened by a growth rate method, and the inhibition rate of the metabolite of the six strains on the A.solani is 47.18-81.92%, wherein the inhibition rate of the fermentation filtrate of the HJG-5 strain is 81.92% which is the highest. On the basis of this experiment, the optimal strain HJG-5 was selected for subsequent studies.

TABLE 2 screening of antagonistic actinomycetes in zanthoxylum bungeanum

Note that the lower case letters in the English letters in the Table indicate the significance of the difference at the P <0.05 level for the new repolarization test.

3.1.3 determination of bacterial inhibition Spectroscopy of HJG-5 Strain

3.1.3.1HJG-5 Strain bacteriostatic activity assay

The bacteriostasis spectrum of HJG-5 strains is measured by taking 12 pathogenic bacteria as indicator bacteria, and Table 3 shows that HJG-5 strains have inhibition effect on tested pathogenic bacteria, and the inhibition rate is 42.87% -76.55%; the inhibition rates of M.laxa and A.solani are the best, and are 77.42% and 76.55% respectively; the bacteriostatic effect on V.mali, S.sclerotomum (lib.) de Bar and C.lindemunianum is poor, and the bacteriostatic effect is 45.98%, 49.84% and 42.87% respectively; the bacteriostasis rate to other pathogenic bacteria is between 51.91 and 70.54 percent, and the effect is shown in figure 1.

TABLE 3 inhibitory Effect of 3HJG-5 strains on 12 pathogenic bacteria

3.1.3.2HJG-5 bacterial strain fermentation filtrate bacteriostasis spectrum determination

The fermentation filtrate of the HJG-5 strain is subjected to bacteriostasis spectrum measurement to ensure the research and development value. As is clear from table 4, the inhibition effects of the strain fermentation filtrates on the 12 germs to be tested are significantly different, and the inhibition rates on m.laxa and a.solani are best at 80.47% and 82.39%, respectively, and the inhibition rates on f.graminearum, l.libanotis and m.fructicola are also better at 73.88%, 75.26% and 70.32%, respectively. The inhibition rate of the inhibitor on R.solani, V.mali and S.sClerotiorum (lib.) debry is poor and is lower than 40%, and the effect is shown in figure 2.

TABLE 4 inhibitory Effect of 4HJG-5 Strain fermentation filtrate on 12 pathogenic bacteria

3.2HJG-5 Strain classification status

3.2.1 morphological characteristics

HJG-5 strain is cultured on oat flour agar medium for 2-3 days, the colony surface is smooth and white, white aerial hyphae grow from the colony edge after 6 days, and gradually turn to light gray after 8 days. The colony morphology is conical uplift in the culture process, the edge of the colony is provided with 4-6 circles of concentric ring lines, and the periphery is provided with a halo-shaped soil edge. Microscopic observation shows that aerial hyphae are developed and have more branches, and hyphae in the substrate have no transverse septa and are not broken, and the effect is shown in figure 3.

3.2.2 culture characteristics

Referring to Table 5, HJG-5 strain grows better on ISP2, PDA and nutrient agar, the colony surface is raised to be compact fluffy powder, aerial hyphae are developed and are white or grey white, and the hyphae in the substrate are brown yellow or yellow brown. Medium growth on ISP5, light gray aerial hyphae and pale yellow ground hyphae. The growth on ISP4, Chachi's and Gao's No. I culture medium is poor, aerial hyphae are thin, mostly in light white gray color, no substrate hyphae, and hardly grow on ISP 3. None of the 8 media tested produced soluble pigments.

TABLE 5 culture characteristics of endophytic actinomycete HJG-5 on different media

Note: "-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +" means vigorous growth

3.2.3 physiological and Biochemical assays

As shown in Table 6, the results of physiological and biochemical tests show that the endogenous antagonistic actinomycete HJG-5 can produce melanin, can liquefy part of gelatin, and can reduce nitrate into positiveSex, starch hydrolysis, milk coagulation and peptonization without producing H₂S, no growth on cellulose. The strain can not utilize starch, xylose, arabinose and fructose, and has better utilization degree on several carbon sources of glucose, maltose, sucrose, lactose, mannitol and glycerol.

TABLE 6HJG-5 physiological and biochemical characteristics of the strains and carbon source utilization

Note: "-": negative; "+": positive for

3.2.4 molecular biology

The primer 27F/1492R is used, HJG-5 strain DNA is used as a template to carry out PCR amplification, and agarose gel electrophoresis is used for detecting PCR products, wherein the PCR products are shown in figure 4, and the bands are clear and single. After sequencing, the sequence length is 1361 bp. Comparing the 16SrDNA sequence with the registered similar 16SrDNA sequence in Genbank, selecting the gene sequence of the strain with higher homology with the sequence, analyzing by MEGA5.1 software, constructing HJG-5 strain phylogenetic tree by adopting a Neighbor-Joining method, and finally, gathering the HJG-5 strain and streptomyces cinnamomocastanenus and AB184588 in the same branch, referring to figure 5, the homology reaches 99 percent, and the genetic relationship is nearest, therefore, the strain HJG-5 is classified as Streptomyces cinnamomyces cinnamomocastanenus.

3.3 fermentation Condition optimization

3.3.1 cell growth Curve

HJG-5 strain growth curve and antibacterial active substance yield change are shown in FIG. 6, the strain rapidly grows in 0-7d, hypha dry weight increases with fermentation time, reaches peak at 7d, and begins to decline at 8-9d as stationary phase, fermentation 10 d.

According to the bacteriostatic rate curve of HJG-5 strain, the yield of bacteriostatic active substances is gradually increased along with the extension of fermentation time, the bacteriostatic effect begins to appear after fermentation for 1d, the maximum value is 85.89% at 7d, the bacteriostatic rate is stable at 8-9d, no significant difference with the maximum value exists, the descending trend begins to appear at 10d, and the self growth of the strain and the yield of the bacteriostatic active substances are basically kept consistent by combining the hypha dry weight curve. 3.3.2 Medium and culture Condition screening

3.3.1 screening of basal Medium

Five basic culture media A to E are adopted, wherein A: a Gaoshi-I liquid culture medium, a millet liquid culture medium, a soybean powder liquid culture medium, a soybean corn flour liquid culture medium, a: and (3) performing fermentation culture on the PD culture medium, and measuring the bacteriostasis rate of the fermentation filtrate to A.solani by adopting a growth rate method after 7 days. As can be seen from FIG. 7, the bacteriostatic activity of the strains in each fermentation medium is significantly different, wherein the bacteriostatic effect of the fermentation filtrate of the E medium is 84.63% best, and 76.27% of the D medium, and the bacteriostatic effect of the A medium is 4.52% worst, so that the E medium, i.e. PD, is selected as the basal medium for the next optimization test.

3.3.2 carbon Source selection

With PD as a basic culture medium and under the condition of adding different C sources, the fermentation filtrate of the HJG-5 strain has obvious difference on the A.solani antibacterial activity, as shown in figure 8, CK is PD, when maltose is used as the C source, the antibacterial activity is the best, 86.29%, glucose CK is 84.28%, mannitol, starch, lactose and corn flour are the second, when sucrose is used as the carbon source, the antibacterial effect is the worst, and is 31.28%. Maltose is therefore the optimal C source.

3.3.3 Nitrogen Source selection

The difference of the bacteriostatic activity of HJG-5 strains from different N sources is shown in FIG. 9, and of the 8 tested N sources, the bacteriostatic activity of the strain under the condition of tryptone nutrition is the best, 87.60%, and the difference is significant compared with CK without nitrogen source with bacteriostatic rate of 84.32%, and then yeast powder and (NH) are used₄)₂SO₄83.64% and 83.52% respectively, beef extract, peptone and KNO₃Compared with soybean powder, the antibacterial activity is less than CK, and when urea is added as a nitrogen source, the antibacterial effect is 19.32% at the worst, so tryptone is selected as an N source in later tests.

3.3.4 orthogonal optimization of fermentation conditions

The basis of the selected optimal medium componentsIn the above, five factors of the rotation speed, the pH, the liquid loading amount, the inoculation amount and the fermentation time are selected to further optimize the fermentation conditions, and the results are shown in table 7, wherein the bacteriostatic effect of the group 5 is 91.30% and the corresponding horizontal combination is A2B1C2D3E4, and from the visual analysis of the trend of fig. 10, the optimal horizontal combination is A4B3C4D3E4, which is not included in the orthogonal design test table, so that the test at the level is added once to increase the accuracy of the test result, and the bacteriostatic rate of the additional test is 91.78% and is higher than that of the group 5. From the range analysis, the influence degrees of the five factors on the bacteriostatic activity of the fermentation filtrate of the HJG-5 strain on the A.solani are the rotating speed in sequence>Liquid loading amount>Time of fermentation>PH>The inoculation amount is that the fermentation condition of HJG-5 strain is 180 r.min after the optimization of orthogonal test^-1The pH is 8, the liquid loading is 125mL/250mL, the inoculation amount is 8%, and the fermentation time is 9 d.

TABLE 7 analysis table of fermentation conditions of 7 HJG-5 strains in orthogonal test

3.3.5 optimized detection of biological Activity

The HJG-5 strain is fermented according to the optimized culture medium formula and culture conditions, the bacteriostatic activity of the fermentation filtrate on A.solani is measured by a growth rate method, and experimental results show that the bacteriostatic activity of the optimized fermentation filtrate is remarkably improved from 83.70% before optimization to 91.78%, see fig. 11.

3.4HJG-5 Strain fermentation filtrate stability assay

3.4.1 thermal stability

HJG-5 strain fermentation liquor is treated at different temperatures for 30min, the bacteriostatic activity is shown in figure 12, the bacteriostatic activity is gradually reduced along with the increase of the temperature, but except that the bacteriostatic activity is reduced to 22.54% after the treatment at 121 ℃, the bacteriostatic activity is more stable within 80 ℃, the bacteriostatic rate is maintained at about 80%, the fermentation liquor treated at 100 ℃ has 63.63% of bacteriostatic rate on A.solani, namely, the bacteriostatic activity is still stronger, and the test result shows that the HJG-5 strain metabolite has better heat stability.

3.4.2 acid-base stability

The change of the bacteriostasis rate of the fermentation liquor after acid and alkali treatment is shown in figure 13. The antibacterial activity is best under the environment of pH7-pH9, the antibacterial activity is not obviously different from that of an untreated CK group, and the antibacterial rate is gradually reduced but is maintained at about 80% along with the increase of the pH value. In conclusion, the bacteriostatic active substance of the strain has stronger pH stability.

3.4.3 ultraviolet radiation stability

The bacteriostatic activity of the fermentation filtrate after being irradiated by ultraviolet light at different times is shown in fig. 14, the inhibition rate of the fermentation broth to a.solani is slightly reduced along with the accumulation of irradiation time, but the bacteriostatic rate is about 80% overall, the bacteriostatic rate after being irradiated for 24 hours is 79.51%, and the bacteriostatic rate is reduced by only 6.5% compared with that of a CK group which is not subjected to ultraviolet treatment, so that the bacteriostatic active substances are stable under ultraviolet irradiation and can not be degraded or changed.

3.4.4 storage stability

At 4 ℃, the fermentation filtrate has better storage stability as shown in fig. 15, within 30 days, the bacteriostatic activity is more than 80%, the bacteriostatic rate is not significantly different at 40d-80d, and is about 77%, and the bacteriostatic rate of the A.solani is still 74.03% and is 85.15% of the bacteriostatic activity of the original fermentation broth at 90 d.

3.5HJG-5 bacterial strain fermentation filtrate with bacteriostatic action

3.5.1 Effect on A.solani hypha morphology

The effect of strain fermentation filtrate on the morphology of a. solani hyphae is shown in fig. 16, where a is normal hyphae; B. c is hypha treated by the strain HJG-5; solani hyphae normally grow, are smooth and uniform in thickness, are clearly spaced, and are uniformly dispersed in intracellular mycelium lysates, and are complete in structure. HJG-5 after fermentation liquid treatment, abnormal hypha growth, abnormal swelling, uneven thickness, cytoplasm aggregation, and extravasation.

3.5.2 Effect on A.solani cell Membrane Permeability

When the permeability of mycelium cell membrane is changed and intracellular substances are infiltrated, the change of conductivity value is caused. Therefore, the influence of the bacteriostatic substance on the permeability of the microbial membrane is often reflected by measuring the conductivity value. Referring to fig. 17, the conductivity values of the treatments tended to increase with time, and the increase was proportional to the amount of fermentation filtrate added, while the conductivity of CK increased very little and the curve was smooth. Within the same time, the conductivity value is in positive correlation with the addition of the fermentation filtrate. Therefore, it can be known that the cell membrane permeability of the tomato early blight bacteria is greatly influenced by the metabolite of the strain, and the damage degree is increased along with the increase of the concentration of the fermentation filtrate and the prolonging of the time.

3.6 determination of growth promotion and biocontrol effects of fermentation filtrate of 3.6HJG-5 Strain

3.6.1 Effect on tomato seed Germination

The GI of the tomato seed is in a situation of rising first and then falling along with the increase of the concentration of the fermentation filtrate, see fig. 18, wherein the fermentation liquor diluted by 20X, 50X and 80X has a significant promoting effect on seed germination, the germination index of the fermentation liquor diluted by 20X after treating the tomato seed is 49.07, which is increased by 16.75% compared with 42.03 of a control, when the dilution multiple is gradually increased, the promoting effect is weakened, the GI of the fermentation liquor diluted by 100 times has no significant difference with the CK, and the fermentation liquor with high concentration after soaking the seed presents a suppression trend on seed germination.

3.6.2 Effect on tomato seedling growth

Referring to fig. 19, after the root irrigation treatment by the fermentation liquids with different concentrations, the growth conditions of the tomato seedlings are that when the concentration of the fermentation liquid is diluted at 100X-20X, the plant heights, the root lengths, the fresh weights and the dry weights of the tomato seedlings increase with the increase of the concentration of the fermentation liquid, particularly the effect of the 20X dilution liquid is the best, the plant heights, the root lengths, the fresh weights and the dry weights of the tomato seedlings are respectively increased by 29.0%, 20.4%, 24.9% and 35.9% compared with the CK group, the growth promotion effect is weakened along with the increase of the concentration of the fermentation liquid, the inhibition effect is gradually enhanced, and the fermentation stock liquid has a significant inhibition effect on various indexes of the plants relative to the CK, so that the fermentation liquid of the strains has a certain promotion effect on the plant growth at low concentration, and restricts the plant growth at high concentration, see fig. 20.

3.6.3 Ex vivo control effect

Prevention effect of 3.6.3.1HJG-5 strain fermentation filtrate on isolated leaf A

HJG-5 strain fermentation liquor on in vitro leaf has effect on A.solani, see Table 8, fermentation liquor with different concentrations on in vitro leaf has effect on A.solani with different degrees, tomato leaf of CK becomes infected after 1 day of inoculation of pathogenic bacteria, and lesion spot area gradually expands to 485.10mm after 4 days³The leaf control effect of spraying the stock solution of the fermentation liquor is the best, and the lesion area is only 122.33mm³The inhibition rate is 80.60%, when the dilution factor of the fermentation liquor is increased, the control effect is gradually reduced, and the control effect difference with the fermentation stock solution is obvious, see fig. 21.

TABLE 8 prevention and treatment effects of fermentation filtrate of 8HJG-5 Strain on in vitro leaf A

3.6.3.2HJG-5 strain fermentation filtrate has effect in preventing isolated fruit A.solani

The inhibition effect difference of the fermentation filtrates with different concentrations on the in vitro fruits on the a.solani is obvious, see table 9, the lowest prevention and treatment effect is 20X diluted fermentation liquor, the inhibition rate on the a.solani is 44.34%, while the high-concentration fermentation liquor has obvious prevention effect on the a.solani on the in vitro tomato fruits, the fermentation liquor stock solution with the best prevention and treatment effect has the inhibition rate up to 82.96%.

TABLE 9 prevention and control effects of fermentation filtrate of 9HJG-5 strain on isolated fruit A

3.7 preliminary isolation of bacteriostatic active substances and determination of biological Activity

3.7.1 screening of optimal extraction solvent

The method comprises the steps of performing crude extraction on bacteriostatic active substances in HJG-5 strain fermentation filtrate by using 5 organic solvents with different polarities, performing rotary evaporation and concentration, and then determining the bacteriostatic activity of each organic phase and each water phase relative to A.solani, wherein the bacteriostatic activity of each organic phase and each water phase is determined according to the table 10, wherein the five treated organic phases and the five treated water phases have bacteriostatic effects with different degrees, when n-butyl alcohol is used as an extracting agent, the bacteriostatic effect difference between the organic phase and the water phase is the largest, the bacteriostatic rate of the organic phase is 93.12%, and the bacteriostatic rate of the water phase is 43.39%. With the reduction of the polarity of the organic solvent, the antibacterial activity of the organic phase of each extracting agent is gradually reduced, and the antibacterial activity of the petroleum ether extract is only 30.03 percent, which shows that the active substance is a fat-soluble substance with larger polarity, so the n-butyl alcohol is selected as the optimal extracting agent.

TABLE 10 bacteriostatic activity of different organic solvent extracts of strain HJG-5

3.7.2 silica gel column chromatography for separating antibacterial active substance

Extracting the fermentation liquor by using n-butanol to obtain an active substance crude extract, loading the sample by using a wet method, performing gradient elution by using petroleum ether, chloroform and methanol in different proportions, collecting 20mL of the active substance crude extract in each tube, performing TlC detection, combining fractions with the same Rf (specific transfer value) to obtain 10 components, performing reduced pressure concentration on the components, and determining the antibacterial activity of the components on A.solani by using an antibacterial circle method, wherein the antibacterial activity is shown in figure 22. The component 8 has the best bacteriostatic effect, the diameter of a bacteriostatic zone is 18.63mm, and the components 4, 5 and 10 have almost no bacteriostatic activity on A.solani.

4 conclusion

4.1 isolation, screening and identification of antagonistic actinomycetes of pepper endophytic

The invention adopts a tissue homogenate method to obtain 11 endophytic actinomycetes from each tissue part of a pepper plant through co-separation, 1HJG-5 strains with good antibacterial activity are obtained through screening by a plate confrontation method and a fermentation broth growth rate method, and the bacterial strains have wider antibacterial spectrum by measuring the antibacterial spectrum, particularly can well inhibit the growth of M.laxa and A.solani, the inhibition rate of viable bacteria to the viable bacteria is more than 70 percent, the fermentation broth has the inhibition rate of more than 80 percent, and the antibacterial activity of metabolites is higher than that of the viable bacteria on the whole, so that the HJG-5 strains can be inferred to generate antibacterial active substances through metabolism to inhibit the growth of pathogenic bacteria and are strains with good biocontrol potential.

Through strain morphological characteristics, culture characteristics, physiological and biochemical identification and strain 16SrDNA sequencing analysis, the strain HJG-5 is identified as the streptomyces cinnamomi, but the strain can utilize lactose, mannitol and glycerol to grow very weakly on Gao synthesized No.1 and Chase and can grow well on PDA, and no soluble pigment can be observed on 8 culture characteristic observation culture mediums used, which is different from the description of streptomyces cinnamomi cinnabanica strain published by Shenmei, on the other hand, the strain HJG-5 is separated from the root of the plant of Zanthoxylum bungeanum, while the streptomyces cinnamomi cina is separated from soil, so that the strain HJG-5 is supposed to be a part of the soil actinomyces originally, and enters the root of the plant after long-term evolution, and the self-physical and chemical properties are changed due to the change of the environment.

4.2 study of fermentation conditions of 4.2HJG-5 Strain

The optimal basal medium PD is screened out from the 5 culture mediums to be tested, and the optimal C source maltose and the optimal N source tryptone are screened out by a single factor method; the culture conditions for screening the most suitable strains by adopting the orthogonal optimization method are as follows: rotation speed of 180r min^-1pH8, liquid loading amount of 125mL/250mL, inoculation amount of 8%, and fermentation time of 9 days. And (3) carrying out biological activity determination on the optimized culture medium formula and the fermentation conditions, and finding that the antibacterial activity of the culture medium formula and the fermentation conditions on the A.solani is obviously improved.

4.3HJG-5 strain metabolite physicochemical properties and bacteriostatic mechanism

In order to explore the physicochemical properties of antibacterial active substances, various stability tests are carried out on strain fermentation liquor, and the results show that the HJG-5 strain metabolite has better thermal stability, the antibacterial rate is maintained at about 80% below 80 ℃, and the strain metabolite is very stable in neutral and weak alkaline environments; the bacteriostatic activity of the bacteriostatic substance is not changed by ultraviolet irradiation; the storage stability is good at 4 ℃, and the inhibition rate of the A.solani at 90 days can reach 85.15% of the antibacterial activity of the original fermentation liquor. The physical and chemical properties of the bacteriostatic active substances generated by the strain are stable.

The invention utilizes an optical microscope to observe the influence of the bacteriostatic active substance of the HJG-5 strain on the growth of the A.solani hypha and simultaneously determines the influence of the bacteriostatic active substance on the permeability of the hypha cell membrane. The results show that after the fermentation liquor of the HJG-5 strain is treated, compared with the hyphae which normally develop, the A.solani hyphae have the phenomena of bending and folding, abnormal swelling, cytoplasm aggregation, extravasation and the like. The conductivity value of the hypha culture solution of the pathogenic bacteria is measured, so that the conductivity value is increased by adding the fermentation solution and is in positive correlation with the concentration of the fermentation solution and the treatment time, namely hypha cell membranes are damaged, and intracellular substances are oozed, which is also consistent with the result of hypha microscopic observation.

4.4 growth promoting and biocontrol effects of 4.4HJG-5 strain fermentation liquor

According to the invention, through the determination of the influence of fermentation liquor of HJG-5 strains with different concentrations on tomato seed germination and seedling growth, the fermentation liquor can promote seed germination and plant growth at a certain concentration, the effect is optimal when diluted by 20X, GI of the treated seeds is increased by 16.75% compared with CK, the plant height and root length of tomato seedlings, fresh weight and dry weight are also obviously increased compared with CK group.

The invention adopts the stab wound inoculation method to measure the in vitro control effect of HJG-5 strain fermentation liquor, and finds that the fermentation filtrate has good bacteriostatic effect on the A.solani on the in vitro leaves and fruits of the tomatoes, and the fermentation stock solution can reach the bacteriostatic rate of more than 80%. The HJG-5 strain has good biological control potential and good application prospect in plant disease control according to the growth promoting capability and biological control capability of the strain.

4.5HJG-5 bacterial strain bacteriostatic active substance preliminary separation and biological activity determination

The invention uses 5 organic solvents with different polarities to carry out crude extraction on the antibacterial active substance in the HJG-5 strain fermentation filtrate, and the result shows that the n-butyl alcohol can furthest extract the antibacterial active substance, namely the active substance is a fat-soluble substance with larger polarity. And (3) after gradient elution separation and TLC detection are carried out on the active substance crude extract obtained by extraction, 10 components are obtained in total, and the bacteriostatic activity of the active substance crude extract on A.solani is determined by a bacteriostatic ring method, wherein the bacteriostatic activity of the component 8 is the best, and the diameter of the bacteriostatic ring is 18.63 mm. The high-efficiency broad-spectrum bacteriostatic activity of the zanthoxylum bungeanum endophytic actinomycete HJG-5 shows that the zanthoxylum bungeanum endophytic actinomycete has strong biocontrol potential.

It should be understood that the above-described specific embodiments are merely illustrative of the invention and are not intended to limit the invention. Obvious variations or modifications which are within the spirit of the invention are possible within the scope of the invention.

Claims (10)

1. An endogenous actinomycete HJG-5 of Zanthoxylum bungeanum, is characterized in that, described HJG-5 bacterial strain is preserved in China Microorganism Culture Collection General Microorganism Center, preservation date: 2017.11.30, preservation registration number is 14969. 2. a kind of Zanthoxylum bungeanum endophytic actinomycetes HJG-5 according to claim 1, is characterized in that, described HJG-5 bacterial strain is classified as Streptomyces cinnamonis versicolor. 3. a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 1, is characterized in that, described HJG-5 bacterial strain is cultivated 2-3d on oat flour agar medium, and the bacterium colony surface is smooth and white, cultivates After the 6th day, white aerial hyphae began to grow from the edge of the colony, and gradually turned light gray after 8d; during the cultivation process, the colony shape was a conical bulge, with 4-6 circles of concentric rings on the edge of the colony. There is a halo-shaped soil edge; through microscopic observation, the aerial hyphae are developed with many branches, and the hyphae in the base have no transverse septum and are not broken. 4. a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 1 and 2, is characterized in that, the culturing characteristic of described HJG-5 bacterial strain is, on ISP ₂ , PDA and nutrient agar, the bacterium colony surface is uplifted Dense velvet powder, developed aerial hyphae, white or grayish-white, and basal hyphae brownish-yellow or yellowish-brown; on ISP5, aerial hyphae are light gray, and basal hyphae are light goose-yellow; on ISP _4. The hyphae on Chashi and Gao's No. 1 medium are thin, mostly pale white-gray, without intrabasal hyphae; almost no growth on ISP ₃ . 5. a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 3, is characterized in that, described HJG-5 bacterial strain can produce melanin, can make part of gelatin liquefaction, nitrate reduction is positive, starch hydrolysis, Milk coagulates and peptonizes, does not produce H ₂ S, does not grow on cellulose; cannot utilize starch, xylose, arabinose and fructose, but can utilize glucose, maltose, sucrose, lactose, mannitol and glycerol. 6. The purposes of a kind of prickly ash endophytic actinomycetes HJG-5 described in claim 1-5 in producing growth-promoting active substances and broad-spectrum bacteriostatic active substances. 7. the purposes of a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 6 in producing growth-promoting active substance, broad-spectrum antibacterial active substance, it is characterized in that, described growth-promoting active substance, broad-spectrum The bacteriostatic active substance is derived from the fermentation broth after the HJG-5 strain is fermented and cultured. 8. the purposes of a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 7 in producing growth-promoting active substance, broad-spectrum antibacterial active substance, it is characterized in that, described HJG-5 bacterial strain fermented and cultivated. The culture conditions are: rotating speed 180r·min ^-1 , pH=8, filling volume 125mL/250mL, inoculum volume 8%, culture temperature 28°C, and culture time 9d; the carbon source used in the fermentation medium is maltose, and the nitrogen source is Tryptone, and the ratio of maltose:tryptone is 2:1. 9. the purposes of a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 8 in producing growth-promoting active substance, broad-spectrum antibacterial active substance, it is characterized in that, described fermented liquid has better heat Stability, the bacteriostatic rate is maintained at about 80% below 80 ℃, and it is very stable in neutral and weak alkaline environments; the bacteriostatic activity does not change after UV irradiation; under the condition of 4 ℃, the storage stability is good. The inhibition rate of A.solani can reach 85.15% of the bacteriostatic activity of the original fermentation broth. 10. the purposes of a kind of prickly ash endophytic actinomycetes HJG-5 according to claim 9 in producing growth-promoting active substance, broad-spectrum antibacterial active substance, it is characterized in that, described fermented liquid is diluted 20 times more than fruit Preferably, the germination index of tomato seeds is increased by 16.75% after the 20-fold diluted fermentation broth; the plant height, root length, fresh weight and dry weight of tomato seedlings are increased by 29.0%, 20.4%, 24.9% and 35.9% respectively.

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