CN108165506A - A kind of golden yellow streptomycete and its application - Google Patents

Abstract

It is golden yellow streptomycete XJC SDSIM1 the present invention provides a kind of actinomyces are provided, deposit number is CCTCC NO：M 2017491.The golden yellow streptomycete XJC SDSIM1 growths pH ranging from 5 10 of the present invention, growth temperature is 22 32 DEG C, it cannot be grown on culture medium of the NaCl contents more than 5%, with broad-spectrum antibacterial activity, there is good antagonism to Colletotrichum capsici, cucumber fusarium axysporum, Colletotrichum gloeosporioides Penz in Mango, rice blast fungus, fusarium graminearum, Botryosphaeria berengeriana f. sp and Strawberry anthracnose bacterium etc., with wide development space, there is good development prospect.

Description

A kind of streptomyces aureus and application thereof

technical field

The invention belongs to the field of microorganisms, and in particular relates to a Streptomyces aureus and application thereof.

Background technique

An important direction of biopesticide research is to find new substances to control harmful organisms from microbial metabolites. With the needs of environmental protection, green food and agricultural sustainable development, agricultural antibiotics, as low-toxicity and low-residue biopesticides, have been paid more and more attention by people. Currently, nearly 70% of the approximately 22,500 biologically active substances found in microorganisms are produced by actinomycetes. Actinomycetes are the main source of antibiotics, and 60% of known antibiotics are produced by actinomycetes, mainly concentrated in the genus Streptomyces, such as streptomycin, aureomycin, tetracycline, paromomycin, mitomycin and other important antibiotics.

With the in-depth study on the metabolic characteristics of soil actinomycetes and the further improvement of isolation and culture methods, more and more soil actinomycetes will be isolated. Streptomyces has been considered as the main source of various antibiotics. Genetic diversity analysis shows that there are tens of thousands of antibiotics produced by Streptomyces, and only a small part has been discovered so far.

Although actinomycetes were originally used in medicine, they have been widely used in agriculture due to their low impact on the ecological environment and less pollution. Many studies have confirmed that actinomycetes play a significant role in the biological control of plant diseases, insect pests and weeds, and have a good application prospect.

Therefore, it is particularly important to discover new strains with antibacterial activity.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a kind of actinomycetes, which is a kind of Streptomyces aureus, has broad-spectrum antibacterial activity, has good antagonism to various germs, and has broad development space and application prospects.

The first aspect of the present invention is to provide an actinomycete, which is Streptomyces aureus XJC-SDSIM1, and the preservation number is CCTCC NO: M 2017491.

The second aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention in antagonizing capsicum anthracnose, and/or cucumber wilt, and/or rice blast fungus, and/or wheat head blight , and/or apple ringworm, and/or strawberry anthracnose.

The third aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention in the prevention and treatment of pepper anthracnose, and/or cucumber wilt, and/or rice blast, and/or wheat head blight, and/or apple ring spot, and/or strawberry anthracnose.

The fourth aspect of the present invention is to provide the fermentation liquid of actinomycetes or the filtrate of the fermentation liquid as described in the first aspect of the present invention.

The fifth aspect of the present invention is to provide the fermented liquid as described in the fourth aspect of the present invention or the filtrate of fermented liquid in antagonizing capsicum anthracnose, and/or cucumber wilt, and/or rice blast fungus, and/or Or wheat scab, and/or apple ringworm, and/or strawberry anthracnose.

The sixth aspect of the present invention is to provide the fermented liquid as described in the fourth aspect of the present invention or the filter liquid of fermented liquid in preventing and treating capsicum anthracnose, and/or cucumber wilt, and/or rice blast, and/or Use in wheat head blight, and/or apple ring blight, and/or strawberry anthracnose.

The seventh aspect of the present invention is to provide a bacterial agent, which contains the actinomycetes described in the first aspect of the present invention.

The Streptomyces aureus XJC-SDSIM1 (streptomyces sanglieri XJC-SDSIM1) of the present invention has a growth pH range of 5-10, a growth temperature of 22-32°C, cannot grow on a medium with a NaCl content greater than 5%, and has broad-spectrum antibacterial Active, has good antagonistic effect on capsicum anthracnose, cucumber wilt, mango anthracnose, rice blast fungus, wheat scab, apple ringworm and strawberry anthracnose, etc., has broad development space, has good Develop application prospects.

Description of drawings

Figure 1 shows the morphology of sporocydia (A) and spores (B) of strain XJC-SDSIM1.

Figure 2 is the phylogenetic tree of strain XJC-SDSIM1 and related strains constructed based on 16S rDNA sequence.

Detailed ways

The present invention will be further described below in conjunction with specific examples, so as to better understand the present invention.

The invention provides an actinomycete, which is Streptomyces aureus XJC-SDSIM1 (streptomycessanglieri XJC-SDSIM1), the preservation number is CCTCC NO: M 2017491, the preservation date is September 11, 2017, and the preservation unit is China Typical Culture Object Preservation Center, located at Wuhan University, Wuhan, China. The Streptomyces aureus XJC-SDSIM1 of the present invention is isolated from tea tree rhizosphere soil collected in Pu'er City, Yunnan Province.

1 Experimental materials

1.1 Test soil

The rhizosphere soil of tea trees in Pu'er City, Yunnan Province was collected, mixed evenly in a sterile sealed bag, sealed, and stored in an ice box. After collection, remove roots, stones and other sundries, and store them in the refrigerator at 4°C for later use.

1.2 Test medium

The test medium used in this experiment includes isolation medium, culture medium for observation of culture characteristics, physiological and biochemical medium and fermentation medium (Xu Lihua, 2007; Huang Xiaolong, 2009).

Table 1 The isolation medium and formula of actinomycetes

Table 2 culture characteristics observation medium and its formula

Table 3 Medium required for observation of physiological and biochemical characteristics

Table 4 fermentation medium and its formula

1.3 Reagents and equipment used in this experiment

(1) Main reagents

Table 5 Main biochemical reagents and their sources

(2) Instruments and equipment

Table 6 Instruments and Equipment

1.4 Pathogens tested

Fusarium oxysporum f.sp. Cubense, FOC4, is used for the screening of anti-fusarium active actinomycetes and the determination of antibacterial activity. Curvularia fallax, Curvularia lunata, Btortosphaeria dothidea, Colletotrichum acutatum, Colletotrichum gloeosporioides, Strawberry anthracnose ( Colletotrichum fragariae), Botrytis cinerea, Cucumber wilt (Fusarium oxysporum (Schl.) F.sp cucumbererinum Owen), Mango anthracnose (Colletotrichum acutatum), Rice blast (Pyricularia oryae Cav), Fusarium wilt of wheat Disease (FusaHum graminearum Sehw), apple ring disease (Botryosphaeria dothidea) for the determination of antimicrobial spectrum.

2 Experimental methods

2.1 Isolation and identification of actinomycetes in rhizosphere soil

2.1.1 Isolation and purification of rhizosphere soil actinomycetes

Naturally air-dry the soil sample, fully grind it and sieve it, weigh 1g of the soil sample and dissolve it in 10mL of sterile water, heat it at 55°C for 20min and place it on a shaker at 180r/min for 20min to make a suspension. Take the supernatant and dilute it by 10-fold dilution method, prepare soil suspensions of 10 ^-1 , 10 ^-2 and 10 ^-3 , absorb 0.1mL of the suspension and spread it on the separation medium, and invert at 28°C Cultivate for 2-4 weeks, set 3 replicates for each gradient, pick different single colonies and use the streak method for repeated purification on YE medium.

2.1.2 Screening of antagonistic bacteria

Adopt plate confrontation culture method (Sun Jianbo, 2010): Use a puncher with a diameter of 5mm to take the bacteria cake at the edge of the FOC4 pathogen that has been inoculated for 5 days and grow uniformly, and place it in the center of each PDA plate. Inoculate the bacteria to be tested at 2.5cm in the center, inoculate 4 test strains in each plate, take the bacteria inoculated only with FOC4 as a control, place them in an incubator at 28°C and incubate them upside down for 7 days, and observe the results.

2.1.3 Determination of antimicrobial spectrum in the dish

Adopt confrontation culture method (Li Shuzheng, 1997) to carry out antibacterial spectrum measurement to bacterial strain: Utilize the puncher of 5mm to get the bacterium cake of the pathogenic bacterium of 12 kinds of plants that have been purified, and inoculate in the center of PDA flat board, respectively at the distance of pathogenic bacterium A small amount of bacteria to be tested was inoculated on four spots at 2.5 cm of the cake, and the petri dish only inoculated with pathogenic bacteria was used as the blank control group, and each treatment was repeated 3 times. After culturing in the incubator for 4-7 days, the colony growth diameter of the pathogenic bacteria tested was measured by the cross measurement method, and the antibacterial rate was calculated according to the following formula (Xie Ying, 2011; Xia Longsun, 2013):

Colony diameter (mm) = average value of measured colony diameter - 5.0

2.1.4 Observation of culture characteristics of antagonistic strains

Observation of culture characteristics was carried out by referring to the standard medium used in the description of the culture characteristics of actinomycetes in the International Streptomyces Program (Cui BS, 2008). Antagonistic actinomycetes were inoculated on ISP2, ISP3, ISP4, ISP5, ISP6, ISP7 medium, cultured at 28°C for 7-21 days, and the culture characteristics of the strains on each medium were observed and recorded, including colony morphology, The characteristics of the production of aerial hyphae, the color of spores, and the color of hyphae in the substrate.

2.1.5 SEM observation

Soak the coverslip in 0.05g/L potassium dichromate, soak and elute with alcohol, rinse with ultrapure water, blow dry, and sterilize at 121°C for 20 minutes. Insert the sterilized coverslip at 45° on the Gaoshi No. 1 medium connected with actinomycete strains, and culture at 28°C for 7-10 days. Sent to the inspection center for scanning electron microscope observation, put the cover glass sample with bacteria in the vacuum coating machine for gold spray coating, and use the scanning electron microscope to observe the fine structure of the mycelium and spore surface of the strain.

2.1.6 Determination of physiological and biochemical characteristics

Refer to the method of Shirking (1966) and Xu Lihua et al. (2007) to carry out physiological and biochemical identification of the strain, mainly in the following aspects.

(1) Determination of enzymatic properties

①Urease test:

The strain was inoculated on the urease medium, and after culturing at 28°C for 4 days, it was observed whether the medium changed color. Test the ability of the tested strain to produce urease. If the culture medium becomes pink, it is positive, and if it is not white, it is negative.

②Esterase (Tween 20, Tween 80) experiment:

Streak inoculation on esterase medium, culture for 1-2 weeks, and observe every day. Positive if there is a faint halo around its growth, negative if there is no halo.

③Starch hydrolysis:

Nutrient agar was used as the basic medium, and 1.0% soluble starch was added. Inoculate the strains to be tested on a flat plate, and adopt the spot connection method (the diameter of the inoculation should not exceed 5mm). When the strains grow well, add iodine solution around the colonies for detection. If there is a transparent circle around the strain, it means that amylase is produced, and the size of the circle indicates the strength of amylase activity; if no amylase is produced, it is blue.

④ Gelatin liquefaction:

The strains were inoculated on the surface of the gelatin medium without piercing the medium, cultured at 28°C, and the liquefaction degree of the medium was observed on the 5th, 10d, 20d and 30d respectively. Before observation, the test tube needs to be cooled for 20-30 minutes or rinsed with tap water for 30 minutes to observe the liquefaction degree of the medium.

⑤ Cellulose decomposition:

Submerge one end of the filter paper strip in the liquid medium, after sterilization, inoculate the strain to be tested on the filter paper above the liquid level, and observe whether the filter paper strip is decomposed one month later.

⑥ Nitrate reduction:

The strains to be tested were inoculated in nitrate-reducing medium, and cultured at 28°C for 7 and 14 days, and the culture medium without bacteria was used as a control. Add a little culture solution that has been cultivated for 7 days and 14 days respectively in the test tube, add a drop of solution A and solution B, and add dropwise the same for the control. When the solution turns pink, rose red, orange or brown, etc., it is positive for nitrate reduction; if no red appears, add 1 or 2 drops of diphenylamine reagent, if it turns blue, the reduction is negative; if not Blue is still treated as positive.

(2) Single carbon source utilization experiment

In the identification of actinomycetes, one of the important inspection indicators is the utilization of carbon sources. Different actinomycetes have different carbon source utilization capabilities such as sugars, alcohols, organic acids, and fatty acids. Carbon sources used in the experiment: D-fructose, xylose, rhamnose, arabinose, raffinose, melezitose, anhydrous lactose, D-galactose, α-lactose, D-trehalose, D-mannose Sugar, D-ribose, inositol, sorbitol, mannitol, salicin, and soluble starch were added to Pugo's basal medium at a carbon source concentration of 1%. And inoculate the strain to be tested, and culture at a constant temperature of 28°C for 7-14 days. The strain inoculated with the basal medium without adding any carbon source is used as a blank control, and the growth of the strain is observed. If it can grow, it indicates that the strain can utilize this carbon source; if it cannot grow, it indicates that the strain cannot utilize this carbon source.

(3) Single nitrogen source utilization experiment

Nitrogen sources used in the test: histidine, methionine, serine, oxalic acid, glycine, hydroxyproline, phenylalanine, glutamic acid, cysteine, arginine, valine, four Ammonium molybdate hydrate, ammonium acetate, ammonium nitrate, and ammonium sulfate are added to the basal medium at a concentration of 0.5%. Inoculate the strains, culture at a constant temperature of 28°C for 7-14 days, use the strains inoculated with the basal medium without any nitrogen source as the blank control, and observe the growth of the strains. If it can grow, it means that the strain can use the nitrogen source; if it cannot grow, it means that the strain cannot use the nitrogen source.

(4) Determination of some other physiological and biochemical indicators

①Temperature tolerance test:

After inoculating the strain to be tested in the same culture medium, culture them at 20°C, 24°C, 28°C, 32°C, and 36°C for 7-14 days under the condition that other culture conditions are the same, observe and record the growth of the colony conditions to determine the optimum temperature for strain growth.

②pH tolerance test:

The strains to be tested were inoculated in the liquid medium with pH values of 4, 5, 6, 7, 8, 9, and 10, to ensure that other culture conditions were consistent, cultured at 28°C, and observed every other week. Observed until four weeks. Observe and record the growth of the strain each time to determine the optimum pH for the growth of the strain.

③Salt tolerance test:

The strains to be tested were respectively inoculated on different concentrations of NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) medium, and other nutrient components of the medium were all the same. Culture at 28°C, 7 days as an observation period, observe for 4 weeks, and record whether it can grow, so as to determine the upper and lower limit concentrations of NaCl that the strain can tolerate.

④ Hydrogen sulfide production experiment:

Inoculate the strain to be tested on a hydrogen sulfide medium containing a certain proportion of ammonium ions, and culture it at 28°C for 2 weeks. If the medium is black, hydrogen sulfide is produced; if there is no black, there is no hydrogen sulfide production. 2.1.7 Molecular biological identification of antagonistic strains

(1) Extraction of Actinomycetes Genomic DNA

Total DNA was extracted using Bioteke's Bacterial Genomic DNA Rapid Extraction Kit (DP1301, Beijing Bioteke Biotechnology Co., Ltd., China).

(2) Sequencing and analysis of 16S rDNA

①PCR amplification of 16S rDNA:

Genomic DNA of actinomycetes was used as a template, and the general primers 27F and 1492R were used for PCR amplification. The primer sequences are as follows: upstream primer 27F (5'AGAG TTTG ATCC TGGC TCAG 3'), downstream primer 1492R (5'TACG GCTACCTT GTTACGAC TT 3'). The specific reaction system is shown in Table 7, and the reaction procedures (Zhou Junping, 2014; Hao Feifei, 2015; NaYua, 2014) are shown in Table 8.

Table 7 PCR reaction system of 16S rDNA gene

Table 8 PCR amplification reaction conditions of 16S rDNA gene

② Electrophoresis detection of PCR products:

After the PCR reaction, take 5 μL of the PCR amplification product and perform electrophoresis detection on the PCR product of the strain on a 1% agarose gel, and determine whether the connection is successful according to the length of the target fragment.

③Sequencing and building a phylogenetic tree:

The PCR product of the strain was sent to Huada Gene Company for sequence determination. The measured gene sequences were compared using BLAST software, and compared with the known 16S rDNA in GenBank and EzBioCloud databases. The sequences with high homology were found for multiple matching alignment analysis, and the Neighbor-Joining method was used for clustering analysis and phylogenetic tree construction using MEGA5.1 software (Na Yua, 2014; Jianghua, 2015).

2.2 Evaluation of antibacterial activity of antagonistic strains

2.2.1 Determination of plate confrontation broad-spectrum antibacterial activity

The broad-spectrum determination of the strains was carried out using the plate confrontation method: the purified cakes of 12 kinds of plant pathogenic bacteria were taken with a 5mm puncher, and inoculated in the center of the PDA plate, respectively in four places 2.5cm away from the pathogenic bacteria cakes. A small amount of bacteria to be tested was inoculated on the spot, and the petri dish only inoculated with pathogenic bacteria was used as the blank control group, and each treatment was repeated 3 times. After culturing in the incubator for 4-7 days, the colony growth diameter of the tested pathogenic bacteria was measured by the cross method, and the antibacterial rate was calculated according to the following formula (Xie Ying, 2011; Xia Longsun, 2013):

Colony diameter (mm) = average value of measured colony diameter - 5.0

3 Results and Analysis

3.1 Screening and morphological characteristics of actinomycetes

The strains isolated and purified by plate coating were deduplicated according to their colony shape and color on the purified medium, and after the initial screening by the plate confrontation culture method and the re-screening by the Oxford cup method, the strain with the largest inhibition zone was obtained. 1 strain of actinomycetes; numbered XJC-SDSIM1. The morphology of the sporophytes (A) and spores (B) of the strain XJC-SDSIM1 is shown in Figure 1 and Table 9. The basal hyphae of the strain XJC-SDSIM1 are developed and unbroken, and the aerial hyphae are multi-branched; The surface of the spores is smooth.

Table 9 Culture characteristics of strain XJC-SDSIM1 on 6 kinds of media

3.2 Physiological and biochemical characteristics of the strain

Table 10 Some physiological and biochemical characteristics of strain XJC-SDSIM1

+: the result is positive; -: the result is negative.

3.3 Phylogenetic characteristics of strains

The total DNA of the strain XJC-SDSIM1 was extracted, the 16S rDNA sequence of about 1.5kb was obtained by PCR amplification, and its sequence was obtained by sequencing. The sequence information was submitted to EzTaxon for gene sequence similarity search, and a total of 16 strains were obtained that were homologous to the strain XJC-SDSIM1 The sequence information of the highest and named model bacteria was analyzed for phylogenetic analysis, and a phylogenetic tree was constructed (Figure 2). It can be seen from the figure that XJC-SDSIM1 and Streptomyces are clustered together, and the homology of 16S rDNA sequence is in the range of 98.97% to 99.78%. Among them, the homology with Streptomyces sanglieri is the highest at 99.78%. Combined with the results of morphological characteristics, physiological and biochemical characteristics and 16s DNA molecular sequence analysis, the strain XJC-SDSIM1 was preliminarily identified as Streptomyces sanglieri.

3.4 Evaluation of antibacterial activity of strain XJC-SDSIM1

3.4.1 Antagonistic effect of plate confrontation on pathogenic bacteria

The inhibitory effect of table 11 bacterial strain XJC-SDSIM1 on 12 kinds of pathogenic fungi

The data in the table are mean ± standard deviation. Different small letters in the same column indicate significant difference at P<0.05 level by Duncan's new multiple range test.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims (7)

1. An actinomycete, characterized in that it is Streptomyces aureus XJC-SDSIM1, and the preservation number is CCTCC NO: M2017491. 2. actinomycete as claimed in claim 1 is antagonizing capsicum anthracnose bacterium, and/or cucumber wilt bacterium, and/or rice blast fungus, and/or wheat scab, and/or apple ringworm, and /or application in strawberry anthracnose fungi. 3. actinomycete as claimed in claim 1 is controlling capsicum anthracnose, and/or cucumber wilt, and/or paddy rice blast, and/or wheat head blight, and/or apple ring spot, and/or or application in strawberry anthracnose. 4. the filtrate of the fermented liquid of the actinomycetes described in claim 1 or fermented liquid. 5. the filtrate of fermented liquid or fermented liquid as claimed in claim 4 antagonizes capsicum anthracnose bacterium, and/or cucumber wilt bacterium, and/or rice blast fungus, and/or wheat scab, and/or apple The application in the ringworm, and/or the strawberry anthracnose bacteria. 6. the filtrate of fermented liquid or fermented liquid as claimed in claim 4 prevents capsicum anthracnose and/or cucumber wilt and/or paddy rice blast and/or wheat head blight and/or apple round Scabies, and/or Strawberry Anthracnose. 7. A bacterial agent, characterized in that it contains the actinomycetes according to claim 1.