CN113717888B - Streptomyces neoformans and application thereof - Google Patents

Abstract

The invention discloses a streptomyces neoformans YHG-1 with the preservation number of CCTCC NO: m2021007. The streptomycete new species YHG-1 provided by the invention has the growth pH range of 5-8 and the optimal growth pH of 7, can normally grow on a culture medium with the NaCl content of not more than 7%, can produce various enzymes, has antibacterial activity, and has good application prospects in the fields of enzyme industry and agriculture.

Description

Streptomyces neoformans and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to streptomyces neoformans and application thereof.

Background

Streptomyces (Streptomyces) is a gram-positive bacterium with filamentous branched hyphae, has a complex morphological differentiation cycle and strong secondary metabolic capacity, can produce a plurality of metabolites with biological activity, is a microbial resource with huge economic and practical values, and has important values in the fields of agriculture, food, medicine and the like. At present, more than one species of streptomyces are reported, some streptomyces can produce various antibiotics, and some species can produce vitamins, organic acids, alkaloids, enzymes and the like.

Because of the differences in physiological properties and functions among different species in streptomyces, streptomyces is widely used in various fields. The species found in the genus Streptomyces also have thousands of species, and the new species is found again on the basis of the species found in the genus Streptomyces, so that the new species is difficult to find. Therefore, the invention provides a new streptomycete species, which is of great significance for further application of streptomycete.

Disclosure of Invention

In view of the defects of the prior art, the invention provides a new streptomyces and application thereof.

The technical scheme of the invention mainly comprises the following contents:

the invention collects rhizosphere soil of tropical rainforest of Hainan parygo mountain, obtains a new streptomycete strain through separation and purification, and names the new streptomycete strain as streptomycete YHG-1 (streptomycete sp.Nov.YHG-1) and stores the streptomycete strain in China center for type culture Collection in 1 month and 4 days 2021, with the preservation number being CCTCC NO: m2021007, the preservation address is university of Wuhan, china. Through the enzymological characteristic determination of streptomyces YHG-1, the streptomyces YHG-1 can produce esterase, amylase and gelatinase, which shows that streptomyces YHG-1 has good application prospect in the aspects of esterase, amylase and/or gelatinase production.

If Streptomyces YHG-1 is to be cultured, it can be cultured at 25 to 30 ℃ and pH 5 to 8 under conditions containing a nitrogen source, a carbon source and/or sodium chloride.

Preferably, the nitrogen source comprises: histidine, methionine, serine, glycine, cysteine, valine, tyrosine and/or ammonium nitrate.

Preferably, the carbon source comprises: alpha-lactose, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-sorbitol, D-trehalose, L-arabinose, L-phenylalanine, raffinose, melibiose, xylan, D-mannitol, inositol, melezitose, rhamnose, ribose, salicin, soluble starch and/or sucrose.

Preferably, the mass concentration of sodium chloride is not higher than 7%.

On the other hand, the invention also finds that the streptomyces YHG-1 has a certain inhibiting effect on Pyricularia oryzae, and prompts that the streptomyces YHG-1 has a good application prospect in the aspect of preventing and treating Pyricularia oryzae.

The invention has the following effects:

the streptomycete YHG-1 provided by the invention can generate esterase, amylase and gelatinase, which shows that the streptomycete YHG-1 has a certain application prospect in the enzyme preparation industry.

The streptomyces YHG-1 provided by the invention can utilize 8 nitrogen sources such as histidine and 21 carbon sources such as alpha-lactose, and the streptomyces has good utilization capacity of the carbon source and the nitrogen source.

The streptomycete YHG-1 provided by the invention can grow under the condition of high salt and can still normally grow in a culture medium containing 7% of sodium chloride.

The streptomycete YHG-1 provided by the invention has a good inhibition effect on Pyricularia oryzae.

Drawings

FIG. 1 shows the morphology of mycelium (A) and spore (B) of Streptomyces new strain YHG-1 under a scanning electron microscope.

FIG. 2 shows the morphological characteristics of the new Streptomyces species YHG-1 in ISP2-ISP7 medium.

FIG. 3 is a phylogenetic tree of strain YHG-1 and related strains constructed based on the 16S rDNA sequence.

FIG. 4 genome-wide circled map of Streptomyces YHG-1.

FIG. 5 genome-wide ANI alignment of strain YHG-1.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

Example 1 isolation and characterization of Streptomyces neospecies YHG-1

1 materials of the experiment

1.1 test soil

Collecting rhizosphere soil of tropical rainforest of Yingguling in Hainan province, placing in a sterile sealing bag, mixing, sealing, and storing in an ice box. Collecting, removing impurities such as root system and stone, and storing in refrigerator at 4 deg.C.

1.2 test Medium

The test medium of the present experiment included a separation medium, a medium for observing culture characteristics, a physiological and biochemical medium, a fermentation medium, and the like (xuli, 2007; huangxiaolong, 2009).

TABLE 1 isolation Medium for Actinomycetes and its formulation

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TABLE 2 culture characteristic observation culture medium and its formulation

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TABLE 3 culture media for observing physiological and biochemical characteristics

TABLE 4 fermentation media and their formulations

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1.3 reagents and instrumentation used in this experiment

(1) Primary reagent

TABLE 5 major Biochemical reagents and sources

(2) Apparatus and device

TABLE 6 Instrument and apparatus

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2 Experimental methods

2.1 isolation and identification of rhizosphere soil Actinomycetes

2.1.1 isolation and purification of rhizosphere soil Actinomycetes

Weighing 1g of soil sample, dissolving in 10mL of sterile water, and culturing in a shaking table at 180r/min for 20min at normal temperature to obtain a suspension. Taking supernatant, diluting by 10 times dilution method to obtain 10 ^-1 、10 ^-2 And 10 ^-3 The soil suspension is respectively sucked with 0.1mL of suspension and coated on a separation culture medium, inverted culture is carried out for 2-4 weeks at 28 ℃, 3 times of gradient are arranged, different single colonies are selected and repeatedly purified on a YE culture medium by a scribing method.

2.1.2 screening of antagonistic bacteria

Plate confrontation culture (grandson waves, 2010): using a puncher with the diameter of 5mm to take the fungus cakes inoculated with 5d and consistent growth force of the FOC4 pathogenic bacteria edge, inoculating the fungus cakes to the center of each PDA plate, inoculating the bacteria to be tested at a position 2.5cm away from the center of the colony of the pathogenic bacteria, inoculating 4 test strains to each dish, using the culture inoculated with only the pathogenic bacteria as a control, placing the culture in an incubator at 28 ℃ for inverted culture for 7d, and observing the result.

2.1.3 culture characteristic Observation of antagonistic Strain

Observations of the culture characteristics were made by reference to the standard medium used in the international Streptomyces program for the culture characteristics of Actinomycetes (Cui BS, 2008). Inoculating antagonistic actinomycetes on ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 culture media, culturing at 28 deg.C for 7-21d, and observing and recording culture characteristics of the strains on each culture media, including characteristics of colony morphology, aerial hypha production, spore color and intrabasal hypha color.

2.1.4 scanning Electron microscopy

Soaking the cover glass in potassium dichromate with the concentration of 0.05g/L, soaking and eluting by using alcohol, washing by using ultrapure water, blow-drying, and sterilizing for 20 minutes at 121 ℃. The sterilized coverslip was inserted at 45 ℃ onto the culture medium of Goodpasture No. I inoculated with actinomycete strain, and cultured at 28 ℃ for 7-10 days. And (4) conveying the cover glass sample to a detection center for observation of a scanning electron microscope, placing the cover glass sample with the bacteria in a vacuum coating machine for spraying gold coating, and observing the fine structures on the surfaces of hyphae and spores of the strains by using the scanning electron microscope.

2.1.5 measurement of physiological and Biochemical Properties

The physiological and biochemical identification of the strain was carried out by referring to the methods of Shirking (1966) and Xuelihua et al (2007) mainly in the following respects.

(1) Determination of enzymatic Properties

(1) Urease test:

inoculating the strain on a urease culture medium, culturing for 4d at 28 ℃, and observing whether the culture medium is discolored. The test strains were tested for their ability to produce urease, and the medium was positive for pink and negative for no change in color.

(2) Esterase (tween 20, tween 80) experiments:

streaked onto esterase medium for 1-2 weeks, and observed daily. Positive if there is a faint halo around its growth, and negative if there is no halo.

(3) Starch hydrolysis:

nutrient agar is used as a basic culture medium, and 1.0% of soluble starch is added. Inoculating the test strain on a flat plate, adopting a point inoculation method (the inoculation diameter is not more than 5 mm), and when the strain grows well, dripping iodine solution around the bacterial colony for detection. If a transparent circle is generated around the strain, the generation of amylase is indicated, and the size of the circle indicates the strength of the amylase activity; if no amylase is produced, the color is blue.

(4) Liquefaction of gelatin:

the strain was inoculated on the surface of gelatin medium without puncturing the medium, cultured at 28 ℃ and observed for the degree of liquefaction of the medium at 5d, 10d, 20d and 30d, respectively. Before observation, the test tube needs to be cooled for 20-30min or washed by tap water for 30min, and the liquefaction degree of the culture medium can be observed.

(5) Cellulose decomposition:

immersing one end of the filter paper strip in a liquid culture medium, inoculating the strain to be detected on the filter paper sheet above the liquid level after sterilization, and observing whether the filter paper strip is decomposed or not after one month.

(6) Nitrate reduction:

the bacterial strain to be tested is inoculated in a nitrate reduction culture medium and cultured for 7 and 14 days at the temperature of 28 ℃, and the culture medium without inoculation is used as a control. A small amount of culture medium cultured for 7d and 14d was added to the test tube, and one drop of solution A and solution B was added, as was the case with the control. When the solution becomes pink, rose red, orange or brown, etc., the nitrate is positive in reduction; if no red color appears, 1 or 2 drops of diphenylamine reagent is dripped, and if the reagent is blue, the reduction effect is negative; if not blue, it is still treated as positive.

(2) Experiment for Using Single carbon Source

In the identification of actinomycetes, one of the important research indexes is the utilization of carbon sources, and different actinomycetes have different carbon source utilization capacities for sugars, alcohols, organic acids, fatty acids, and the like. Carbon source selected for the test: d-fructose, xylose, rhamnose, arabinose, raffinose, melezitose, anhydrous lactose, D-galactose, alpha-lactose, D-trehalose, D-mannose, D-ribose, inositol, sorbitol, mannitol, salicin, soluble starch and the like are added into a basic culture medium of Pugodi according to the carbon source concentration of 1%. And inoculating the strain to be detected, culturing at constant temperature of 28 ℃ for 7-14d, taking the strain inoculated by a basic culture medium without any carbon source as a blank control, and observing the growth condition of the strain. If the strain can grow, the strain can utilize the carbon source; if the strain can not grow, the strain can not utilize the carbon source.

(3) Experiment for utilization of Single Nitrogen Source

Nitrogen sources selected for the test: histidine, methionine, serine, oxamic acid, glycine, hydroxyproline, phenylalanine, glutamic acid, cysteine, arginine, valine, ammonium molybdate tetrahydrate, ammonium acetate, ammonium nitrate, ammonium sulfate, etc., were added to the basal medium at a concentration of 0.5%. Inoculating strains, culturing at constant temperature of 28 deg.C for 7-14d, taking strains inoculated by basal medium without any nitrogen source as blank control, and observing growth condition of the strains. If the strain can grow, the strain can utilize the nitrogen source; if the strain can not grow, the strain can not utilize the nitrogen source.

(4) Measurement of other physiological and biochemical parameters

(1) Temperature tolerance test:

inoculating the strain to be tested in the same culture medium, culturing at 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C and 36 deg.C for 7-14d under the condition of identical culture conditions, observing and recording the growth condition of bacterial colony, thereby determining the optimum temperature for strain growth.

(2) pH tolerance experiment:

the strains to be tested were inoculated into liquid media with pH values of 4, 5, 6, 7, 8, 9, and 10, respectively, and the other culture conditions were kept consistent, and the culture was carried out at 28 ℃ with observation every other week for four weeks. The growth of the strain was observed and recorded each time to determine the optimum pH for growth of the strain.

(3) Salt tolerance test:

respectively inoculating the strains to be detected on NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) culture media with different concentrations, wherein other nutrient components of the culture media are the same, culturing at 28 ℃, taking 7 days as an observation period, observing for 4 weeks, and recording whether the strains can grow so as to determine the upper and lower limit concentrations of NaCl which can be tolerated by the strains.

(4) Experiment for production of hydrogen sulfide:

inoculating a strain to be detected on a hydrogen sulfide culture medium containing ammonium ions in a certain proportion, and culturing for 2 weeks at 28 ℃, wherein if the culture medium is black, hydrogen sulfide is generated; and if no black color exists, no hydrogen sulfide is generated.

2.1.6 molecular biological identification of antagonistic strains

(1) Extraction of actinomycete genome DNA

Total DNA was extracted using biotek's kit for rapid extraction of bacterial genomic DNA (DP 1301, beijing Baitach Biotech Co., ltd., china).

(2) Sequencing and analysis of 16S rDNA

(1) PCR amplification of 16S rDNA:

PCR amplification was performed using actinomycete genomic DNA as a template and universal primers 27F and 1492R. The primer sequences are as follows: the upstream primer 27F (5-. The specific reaction system is shown in Table 7, and the reaction procedures (Zhoujun nu, 2014; hemiflu, 2015 Na Yua, 2014) are shown in Table 8.

TABLE 7 PCR reaction System for 1696 rDNA Gene

TABLE 8 PCR amplification reaction conditions for 1696 rDNA Gene

(2) And (3) electrophoretic detection of PCR products:

after the PCR reaction is finished, 5 mu L of PCR amplification product is taken to carry out electrophoresis detection on the PCR product of the strain on 1% agarose gel, and whether the connection is successful is determined according to the length of the target fragment.

(3) Sequencing and constructing a phylogenetic tree:

the PCR product of the strain was sent to Huada Gene Co for sequencing. The determined gene sequences were aligned using BLAST software and compared for homology to the known 16S rDNA in GenBank and ezbiocoud databases. Sequences with higher homology are found out for multiple matching array analysis, clustering analysis and phylogenetic tree construction are carried out by using MEGA5.1 software through a Neighbor-Joining method (Na Yua,2014, jianghua, 2015).

(3) Whole genome analysis of strains

The bacterial genome data analysis is carried out by Meiji biology, inc., and the specific method is as follows: a DNA sample qualified for quality detection is inserted into a 400bp fragment by utilizing the currently most widely used second-generation sequencing platform Illumina Hiseq multiplied by 10 to carry out PE150 (pair-end) sequencing, namely double-end sequencing, single-end sequencing reads 150bp in length, and each sample provides a raw sequencing data volume (raw data) which is not less than 100 times of the genome coverage depth. Splicing multiple Kmer parameters of the optimized sequence after the second-generation sequencing by using short sequence assembly software SOAPdenovo2 (http:// soap. Genomics. Org. Cn /), obtaining an optimal contigs assembly result, then comparing reads to the contigs, and carrying out local assembly and optimization on the assembly result according to the relation between the paired-end and overlap of the reads to form scaffolds, wherein basic data such as GC content, sequencing depth and the like can be obtained after the assembly is finished.

(4) Strain genome ANI alignment

The ANI comparison value of the bacteria is the key data of the current bacterial species, so on the basis of obtaining the phylogenetic tree of 16S rRNA and the genome information of the strains, the strains with the nearest genetic relationship with the strains to be detected are determined through the phylogenetic tree of 16S rRNA, the whole genome data of the strains with the nearest genetic relationship is downloaded in a GenBank or EzBioCloud database, and the genome comparison is carried out through an ANI comparator comparison tool of https:// www. If the comparison result is less than the threshold of 95%, the new species can be determined.

3 results and analysis

3.1 screening and morphological characterization of Actinomycetes

Carrying out plate coating separation and streaking on the purified strains, removing the weight according to the colony morphology and the color of the strains on a purification culture medium, and carrying out primary screening and secondary screening by a plate opposite culture method to obtain 1 strain of actinomycetes with the largest inhibition zone; the number is YHG-1.

TABLE 9 cultivation characteristics of Strain YHG-1 on 6 media

3.2 physiological and Biochemical characteristics of the strain YHG-1

TABLE 10 part physiological and biochemical characteristics of Strain YHG-1

Positive result; negative result.

3.3 phylogenetic characteristics of the strains

Extracting total DNA of the strain YHG-1, obtaining a 16S rDNA sequence of about 1.5kb through PCR amplification, sequencing to obtain the sequence, submitting sequence information to EzTaxon for gene sequence similarity search to obtain 9 strains with the highest homology (97.39% -98.29%) with the strain YHG-1 and sequence information of a named model bacterium in total, carrying out phylogenetic analysis, and constructing a phylogenetic tree (figure 3). As can be seen from the figure, the evolutionary relationship of Streptomyces roseififaciens is the closest. As can be seen from FIG. 5, the genome size of strain YHG-1 was 8.71Mb, 431 scfolds were assembled, and the G + C content was 70.764%. The ANI value of the strain is 80.13 percent and is far lower than the threshold value of 95 percent compared with the strain of Streptomyces roseififaciens which is closest to the strain, so that the strain YHG-1 can be judged to be a new species of Streptomyces.

Example 2 evaluation of bacteriostatic Activity

1 method of experiment

Evaluation of antibacterial activity by plate confrontation

The broad-spectrum antibacterial activity of the strain is measured by adopting a plate confronting method: and (3) taking a purified bacterial cake of 7 plant pathogenic bacteria by using a 5mm puncher, inoculating the bacterial cake to the center of a PDA (personal digital assistant) flat plate, respectively inoculating a small amount of bacteria to be detected at four points 2.5cm away from the bacterial cake of the pathogenic bacteria, taking a culture dish only inoculated with the pathogenic bacteria as a blank control group, and repeating the treatment for 3 times. And culturing the strain in an incubator at 28-30 ℃ for 4-7 d, and measuring and recording the width of the bacteriostatic band between the strain to be detected and the pathogenic bacteria mycelium.

2 results

The inhibition rate of the strain YHG-1 on rice blast is 69.4%.

The present invention is not limited to the above-described preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (7)

1. The streptomyces neocatensis YHG-1 is preserved in the China center for type culture Collection in 2021, 1 month and 4 days, and the preservation number is CCTCC NO: m2021007, latin nameStreptomyces sp. Nov. 。

2. The use of the new streptomyces YHG-1 of claim 1 for esterase, amylase and/or gelatinase production.

3. The method for culturing the Streptomyces neocatensis YHG-1 of claim 1, wherein the Streptomyces neocatensis YHG-1 is cultured at a temperature of 25 to 30 ℃ and a pH of 5 to 8 in the presence of a nitrogen source, a carbon source and/or sodium chloride.

4. The culture method according to claim 3, wherein the nitrogen source is: histidine, methionine, serine, glycine, cysteine, valine, tyrosine and/or ammonium nitrate.

5. The culture method according to claim 3, wherein the carbon source is: alpha-lactose, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-sorbitol, D-trehalose, L-arabinose, L-phenylalanine, raffinose, melibiose, xylan, D-mannitol, inositol, melezitose, rhamnose, ribose, salicin, soluble starch and/or sucrose.

6. The culture method according to claim 3, wherein the concentration by mass of sodium chloride is not higher than 7%.

7. The use of the novel streptomyces YHG-1 of claim 1 for controlling rice blast.

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