CN113234642B - Streptomyces thioluteus St-79 and application thereof - Google Patents
Streptomyces thioluteus St-79 and application thereof Download PDFInfo
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Abstract
The invention discloses a Streptomyces thioluteus (Streptomyces thioluteus) St-79 with a preservation number of CCTCC NO: m2020962. The application of the Streptomyces thioluteus (Streptomyces thioluteus) St-79 strain is as follows: promoting the germination of rice seeds, preventing and treating rice bacterial leaf blight and inhibiting plant pathogenic bacteria.
Description
Technical Field
The invention belongs to the field of microbial biological control, and relates to a Streptomyces thioluteus (St-79) strain for antagonizing rice bacterial leaf blight and application thereof in promoting rice seed germination.
Background
The bacterial blight of rice occurs first in japan and the disease occurs in large rice regions in asia due to the spread of rice seeds, which is severe in japan, china, india, etc. At the present stage, the areas affected by bacterial leaf blight in China are mainly concentrated in rice production areas such as east China and China, but are rarely seen in southwest areas, northwest areas and the like. The germs spread through water flow and invade rice through pores or wounds on the leaves, and generally cause disease prevalence in the years of heavy typhoon rainstorm or flooding.
Bacterial blight of rice is caused by Xanthomonas oryzae (Xanthomonas oryzae pv. Oryzae, xoo) and the typical symptoms at the onset are: the yellow-green spots are developed at the leaf apex and the edge of the diseased rice plant, then the diseased rice plant develops into a wavy yellow-green or gray-green symptom, and the spots are expanded into strip spots along the veins, so that the whole leaf can be reached, and the boundary between the diseased part and the healthy part is obvious. At present, the method for preventing and controlling the bacterial blight of rice is mainly chemical prevention and control, but the chemical prevention and control has obvious limitations and harmfulness, and no particularly effective pesticide can be applied to the prevention and control of bacterial diseases at present. Actinomycetes are an important class of biocontrol microorganisms widely used in agricultural production. High MAN Ni and the like (2019) separate actinomycetes from ginkgo forest soil, and the inhibition rate of the actinomycetes on apple canker and apple alternaria leaf spot is measured by a growth rate method to reach more than 98 percent, and the inhibition rate on other pathogenic bacteria such as apple brown spot pathogen, wheat scab pathogen, rice blast and the like is more than 60 percent. Zhanjun et al (2014) screened out No. 12 strains with obvious antagonistic action on rice bacterial blight from 88 strains separated and purified from different environments, and identified as Streptomyces gramineus by morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis. The diameter of the inhibition zone is 60.9mm, and the microbial pesticide has certain application value and development prospect when being used as the microbial pesticide.
In recent years, many scholars have been continuously screened to obtain different kinds of antagonistic actinomycetes, and reports on the control of different plant diseases are insufficient.
The invention of CN101735961A, namely an actinomycete strain and application thereof in preparing aromatic hydroxylamine, relates to an actinomycete (Streptomyces thioluteus) strain: CGMCC No.2725 and application thereof in reducing nitroaromatic compounds to obtain corresponding aromatic hydroxylamine compounds.
Disclosure of Invention
The invention aims to solve the problem of providing Streptomyces thioluteus (St-79) and application thereof in preventing and controlling rice bacterial leaf blight and promoting rice seed germination.
In order to solve the technical problem, the invention provides a Streptomyces thioluteus (St-79) with a preservation number of CCTCC NO: m2020962.
The invention also provides application of the Streptomyces thioluteus (Streptomyces thioluteus) St-79 strain in promoting rice seed germination, preventing and treating rice bacterial leaf blight disease and inhibiting plant pathogenic bacteria (strain fermentation filtrate can inhibit plant pathogenic bacteria).
As an improvement of the use of the present invention:
the dilution of the strain fermentation filtrate promotes the germination of rice seeds; the strain fermentation filtrate can inhibit plant pathogenic bacteria, such as Xanthomonas oryzae pv. Oryzicola, xanthomonas oryzae pv. Oryzae, xanthomonas campestris (Xanthomonas campestris pv. Glycines) and Xanthomonas campestris (Xanthomonas campestris pv. Campestris).
Aiming at the problem that the pesticide control of the rice bacterial blight becomes increasingly exposed, the biological control of the rice bacterial blight by using the microorganisms and the secondary metabolites thereof becomes a research direction. The invention aims to provide 1 Streptomyces thioluteus (Streptomyces thioluteus) St-79 strain with high antagonism to rice bacterial blight and application thereof.
The strain St-79 of the invention is deposited as Streptomyces thioluteus St-79, and the deposited unit is as follows: china center for type culture Collection, collection address: wuhan university in Wuhan, china, the preservation number: CCTCC NO: m2020962, preservation time 2020, 12/24.
The St-79 strain to which the present invention relates is selected from the rhizosphere soil of Pseudolarix amabilis. And identifying the Streptomyces thioluteus according to morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis.
The fermentation filtrate of the St-79 strain of Streptomyces thioluteus has obvious inhibition effect on rice bacterial blight and can be used for preventing and treating rice bacterial leaf blight and the like. St-79 strain is fermented in Gao's No. I liquid culture solution for 6d (at 28 ℃), 0.22 μm filter membrane is filtered to obtain fermentation filtrate, 200 μ L Oxford cup method is used to determine the inhibition effect of the strain on rice bacterial blight (Xanthomonas oryzae pv. Oryzae, xoo), and the diameter of inhibition zone can reach 64 +/-1.2 mm (figure 5). The results of the antibacterial spectrum test show that: the fermentation filtrate of Streptomyces St-79 strain has strong inhibitory effect on 3 representative plant pathogenic bacteria such as rice bacterial streak germ (Xanthomonas oryzae pv. Oryzae), xanthomonas campestris (Xanthomonas campestris pv. Campestris) and soybean bacterial macula (Xanthomonas axonopolis pv. Glycines) etc. (see Table 3). The potted plant control effect test shows that: the relative prevention effect of the St-79 strain fermentation filtrate on the bacterial leaf blight of rice can reach over 88 percent, and the inhibition rate of scab can reach 96.23 percent (see table 4 and figure 8). The streptomycete St-79 strain can provide an excellent strain for developing microbial pesticides, and has a good application prospect in biological control of diseases of crops such as rice, soybean, wild rape and the like.
In summary, 140 actinomycetes are separated from different environment soils, and 1 antagonistic strain St-79 with strong inhibition effect on rice bacterial blight (Xanthomonas oryzae pv. Oryzae, xoo) is obtained through primary screening and secondary screening, wherein the strain is selected from rhizosphere soil of Pseudolarix amabilis. So as to provide a new antagonistic actinomycete resource for the biological control of the rice bacterial leaf blight.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a colony morphology of 10 representative pure strains of actinomycetes (Gao's No. one medium, 7 d);
FIG. 2 shows the colony and microscopic morphological characteristics of the strain of Streptomyces St-79 (Gao's medium No. 7 d);
FIG. 3 shows the results of the physiological and biochemical experiments of the strain of Streptomyces St-79;
FIG. 4 is a tree of Streptomyces St-79 strain evolutions constructed based on 16S rDNA sequences;
FIG. 5 shows the inhibition effect of fermentation filtrate of Streptomyces St-79 strain on the growth of rice bacterial blight;
FIG. 6 is an electron micrograph showing the effect of fermentation filtrate of Streptomyces St-79 strain on rice bacterial blight cells;
in fig. 6, a: treating with a phosphate buffer solution; b: treating fermentation filtrate of the streptomycete St-79 strain;
FIG. 7 is an antibiogram of the strain Streptomyces St-79;
FIG. 8 shows the results of experiments on the prevention of bacterial blight of rice using Streptomyces St-79 fermentation filtrates;
in FIG. 8, A, B, C represent three replicates of the experiment;
CK 1 : blank control group; CK (CK) 2 : xoo treatment control; t is 1 : spraying St-79 strain fermentation filtrate diluted by 4 times after 6h of Xoo pathogen inoculation; t is 2 : spraying St-79 strain fermentation filtrate diluted by 5 times after 6h of Xoo pathogen inoculation; t is 3 : spraying St-79 strain fermentation filtrate diluted by 6 times after 6h of Xoo pathogen inoculation; t is 4 : spraying St-79 strain fermentation filtrate diluted by 4 times after cutting leaves, and inoculating Xoo pathogen after 6 h;
FIG. 9 shows the effect of Streptomyces St-79 fermentation filtrate on rice seed germination.
Detailed Description
The following examples illustrate the invention in detail: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
Example 1 screening and identification of Streptomyces thioluteus (Streptomyces thioluteus) St-79 Strain
1 Strain
(1) Actinomycete strain: separating, purifying, screening and identifying the soil from different habitats, and preserving actinomycete strains.
(2) Rice bacterial blight (Xanthomonas oryzae pv. Oryzae, xoo, hereinafter abbreviated as Xoo) was provided for storage by the laboratory.
2 culture Medium
(1) Solid culture medium No. kao: soluble starch 20g, KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 0.01g of O, 20g of agar and 1000mL of water, and the pH value is 7.4. Is used for the separation, purification and identification of actinomycete strains.
(2) Gao's No. one liquid medium: the same formula (1) as above is used for liquid fermentation culture of actinomycete strains without adding agar.
(3) NA culture medium: 3g of beef extract, 5g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH 7.2. Used for culturing Xoo pathogen.
(4) NA liquid medium: the same formula (3) was used for Xoo activation without agar.
3 Experimental methods
3.1 isolation, purification and preservation of Actinomycetes strains
Separating by soil gradient dilution coating method. Taking 1g of a dry soil sample in 99mL of sterile water, and oscillating the dry soil sample on a 120r/min shaking table for 30min. Supernatant was diluted in gradient (10) -2 ～10 -5 ) Selecting a suitable concentration (10) -4 ) mu.L of the suspension was spread evenly on a Gao's No. one medium plate containing 50. Mu.g/mL potassium dichromate, and incubated at 28 ℃ in an incubator for 7 days until colonies appeared. And (3) selecting a single colony with typical actinomycete colony characteristics, transferring the single colony to another Gao's first culture medium plate, and performing streaking purification for 3 times to obtain a pure strain. The pure strains are numbered and stored in a refrigerator at 4 ℃ for later use.
3.2 screening of antagonistic Xoo Actinomycetes strains
3.2.1 preliminary screening by Co-culture method
Xoo is inoculated in NA liquid culture medium, when shaking table activation culture (160 r/min,28 ℃) is carried out until OD600=0.6, 100 mu L of bacterial liquid is uniformly coated on the NA culture medium to prepare a bacterial-containing plate for standby. Respectively streaking and inoculating the collected actinomycete strains on a Gao's first culture medium plate, culturing at constant temperature of 28 ℃ for 4d, taking actinomycete cakes with the diameter of 5mm by using a sterile puncher, placing the actinomycete cakes in the middle of the Xoo-containing plate, culturing at 28 ℃ for 48h, and observing the inhibition zone. Actinomycete strains with antagonism are preliminarily screened according to the existence and the size of the inhibition zone, and are preserved by 25% glycerol.
3.2.2 Oxford cup method rescreening
(1) Fermentation culture of actinomycete strains: inoculating actinomycetes with antagonistic action into a Gao's first culture medium plate, culturing at 28 ℃ for 3d, punching a hole on the actinomycetes plate by using a sterile puncher with the diameter of 5mm to obtain a bacterium block, inoculating the bacterium block into a 250mL triangular flask containing 50mL of Gao's first liquid culture solution, culturing at 160r/min and 28 ℃ for 6d in a shaking table, centrifuging 50mL of fermentation liquid at 12000r/min for 10min, and filtering with a 0.22 mu m filter membrane to obtain a fermentation filtrate for later use.
(2) Culturing of Xoo: inoculating Xoo into NA liquid culture medium, performing shake bed activation culture (160 r/min,28 deg.C), and allowing bacteria density to reach OD 600 When =0.6, 100 μ L of the bacterial solution was uniformly applied to the NA solid medium.
200 mu L of fermentation filtrate of actinomycete strains is taken, and the inhibition effect of each actinomycete strain fermentation liquid on Xoo is measured by an oxford cup method. Selecting actinomycete strain-St-79 strain with strong antagonistic action according to the size of the inhibition zone.
3.2.3 scanning Electron microscope Observation of morphological changes of Xoo cells after treatment of fermentation filtrate of target Strain
(1) Culturing of Xoo: inoculating Xoo into NA liquid culture medium, performing shake bed activation culture (160 r/min,28 deg.C) until the density of bacteria is OD 600 ＝0.6。
(2) Fermentation culture of actinomycete strains: the fermentation filtrate of actinomycetes having antagonistic action was obtained by the method of 3.2.2 (1).
(3) And (3) observing by a scanning electron microscope: shaking culture of 1mL of the fermentation filtrate and 9mL of Xoo bacterial liquid for 4h (160 r/min,28 ℃), adding phosphoric acid buffer solution treated Xoo bacterial liquid as a control group, centrifuging at 12000r/min for 10min after 4h, and collecting the thallus. Gently washing the collected thalli for 3 times by using phosphate buffer solution, fixing the thalli in a refrigerator at 4 ℃ overnight by using 2.5% glutaraldehyde, and then soaking and cleaning the thalli for three times by using the phosphate buffer solution for 10 minutes each time; dehydrating with 30%, 50%, 70%, 80%, 90%, 95% ethanol for 15min each time, and dehydrating with 100% ethanol for 2 times; soaking the mixture in tertiary butanol for three times, each time for 10 minutes, and then putting the mixture into a vacuum freeze dryer for vacuum drying; and (5) carrying out scanning electron microscope observation after the sample is sprayed with gold.
3.3 identification of antagonistic Streptomyces Xoo St-79 strains
3.3.1 morphological feature Observation
Taking St-79 strain 1 fungus cake (diameter 5 mm), inoculating on Gao's first culture medium plate, activating and culturing for 3d; inoculating 1 bacterial cake (diameter 5 mm) on new Gao's No. I culture medium plate, culturing at 28 deg.C for 7d, and observing the characteristics of colony size, morphology, texture, surface, color, etc. St-79 strain is cultured by the insert method and the embedding method, and morphological characteristics of intrabasal hyphae, aerial hyphae, spore silk and the like of the St-79 strain are observed under an optical microscope and photographed.
3.3.2 physiological and Biochemical characteristic experiments
St-79 strain was subjected to physiological and biochemical tests such as starch hydrolysis, gelatin liquefaction, utilization of carbon source and nitrogen source, and the like.
3.3.3 16S rDNA sequence analysis
Extracting the genome DNA of the St-79 strain, amplifying a 16S rDNA sequence, and sending the sequence to Shanghai bioengineering company for sequencing. Submitting the 16S rDNA sequence obtained by sequencing to GenBank, comparing by using BLAST, analyzing, and establishing a phylogenetic tree by adopting a Neighbor-Joining method in MEGA-X software to determine the type of the actinomycetes.
4 results of the experiment
4.1 obtaining pure strains of Actinomycetes
A total of 140 pure strains of actinomycetes were isolated and purified from soil samples of different environments by isolation and purification, and colonies of 10 representative pure strains of actinomycetes cultured in the No. 7d high-count medium are shown in FIG. 1.
4.2 screening of antagonistic Xoo Actinomycetes strains
The 140 actinomycete strains are subjected to primary screening and secondary screening of Xoo inhibition effect by using a co-culture method and an Oxford cup method, and the Xoo inhibition effect of different actinomycete strains is greatly different. 1 actinomycete strain with good rice bacterial blight antagonism effect is obtained through screening, the number is St-79 strain, the diameter of the inhibition zone measured by Oxford cup method reaches 64 +/-1.2 mm (figure 5), and the St-79 strain is separated from rhizosphere soil of Pseudolarix amabilis.
4.3 Change in morphology of Xoo cells after treatment of fermentation filtrate of St-79 Strain
The observation result of the scanning electron microscope shows that: after the Xoo cells are treated by the fermentation filtrate of the streptomyces St-79 strain, the content of the Xoo cells leaks out, and the thalli are dissolved, so that the fermentation filtrate of the streptomyces St-79 strain has strong lethal effect on the Xoo cells (figure 6).
4.4 identification results of St-79 Strain
St-79 Strain morphological characteristics: culturing on Gao's No. I culture medium at 28 deg.C for 7d to obtain large colony, pink colony and velvet surface; aerial hyphae differentiate to form spore hyphae, which are in primary or secondary rotation (fig. 2).
The physiological and biochemical test results show that: st-79 strain can hydrolyze starch, cellulose, fat, gelatin (Table 1, FIG. 3); various carbon sources can be used, and glucose, mannitol, lactose and the like can be used with better utilization effect (Table 2); a variety of nitrogen sources can be utilized, preferably the nitrogen source is peptone, (NH 4) 2 SO 4 Lysine, histidine; the pH value is 7.3, the optimum rotating speed is 160r/min, and the suitable culture temperature is 28 ℃.
TABLE 1 results of physiological and biochemical tests of Streptomyces St-79 strains
Note: the + + indicates a strong ability, the + indicates a weak ability, and the-indicates no.
TABLE 2 results of carbon source and nitrogen source utilization test of Streptomyces St-79 strain
Note: , + + + + indicates vigorous growth, + + indicates good growth, + indicates general growth, -indicates no growth.
The nucleotide sequence for 16S rDNA sequence analysis is shown in SEQ ID NO 1.
The evolutionary distance from Streptomyces thioluteus (fig. 4) is the closest, with similarity of 99.9%. According to the morphological characteristics, the physiological and biochemical test results and the 16S rDNA sequence analysis of the St-79 strain, and by combining a Streptomyces species-dividing search table and an evolutionary tree analysis, the St-79 strain is identified as the Streptomyces thioluteus.
The deposit information of the strain St-79 of the invention is as follows: the preservation name is Streptomyces thioluteus St-79, and the preservation unit is as follows: china center for type culture Collection, collection Address: wuhan university in Wuhan, china, the preservation number: CCTCC NO: m2020962, preservation time 2020, 12/24.
Example 2 antimicrobial Spectrometry of Streptomyces thioluteus (Streptomyces thioluteus) St-79 Strain
1 Strain
(1) S. thioluteus (Streptomyces thioluteus) St-79 strain.
(2) 4 representative plant pathogenic bacteria: such as 4 representative plant pathogenic bacteria (see Table 3) of rice bacterial streak germ (Xanthomonas oryzae pv. Oryzae), xanthomonas campestris (Xanthomonas campestris pv. Campestris), tomato bacterial leaf spot (Pseudomonas syringae pv. Tomato), and soybean bacterial macula (Xanthomonas axonodis pv. Glycines) for antibacterial spectrum determination of St-79 strain fermentation filtrate.
2 culture Medium
(1) NA liquid medium: beef extract 3g, peptone 5g, naCl 5g, water 1000mL, pH 7.2 (NA solid medium plus agar 20 g). Used for culturing plant pathogenic bacteria.
(2) Gao's No. one liquid medium: soluble starch 20g, KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 O0.01 g and water 1000mL, and the pH value is 7.2-7.4. Seed solutions and methods for St-79 StrainAnd (5) culturing fermentation liquor.
3 Experimental methods
3.1 culture of St-79 Strain and preparation of fermentation filtrate
St-79 strain 1 cake (diameter 5 mm) was taken with a punch, inoculated into Gao's No. one liquid medium, and shake-cultured at constant temperature of 28 ℃ for 72 hours in a liquid volume of 50/250mL, initial pH 7.3, and initial pH 160r/min to serve as seed liquid. Inoculating the seed solution into a Gao's No. I liquid culture medium according to the inoculation amount of 4%, and performing shake fermentation culture for 6d to obtain St-79 strain fermentation liquor. Centrifuging at 12000r/min for 10min in a 50mL centrifuge tube, filtering the supernatant with a 0.22 μm filter membrane, and removing residual spores to obtain fermentation filtrate for later use.
3.2 activation of plant pathogenic bacteria
4 kinds of pathogenic bacteria preserved at 4 ℃ were inoculated into NA solid medium, and activated and cultured at 28 ℃ for 2d. Then transferred to a new NA solid culture medium and cultured for 2d at 28 ℃ for bacterial antimicrobial spectrum determination.
3.3 determination of the antibacterial Spectrum of the St-79 Strain
Respectively inoculating activated 4 plant pathogenic bacteria in NA liquid culture medium, shake culturing (160 r/min,28 deg.C), and allowing bacteria density to reach OD 600 When =0.6, 100 μ L of the bacterial solution was uniformly applied to the NA solid medium. Taking another fermentation filtrate of 200 mu LSt-79 strain, and determining the inhibition effect of the St-79 fermentation filtrate on 4 plant pathogenic bacteria by an Oxford cup method.
4 results of the experiment
The results of the antibiogram measurement of 4 representative plant pathogenic bacteria show that: the streptomyces St-79 strain fermentation filtrate has strong inhibition effect on 3 plant pathogenic bacteria, namely rice bacterial streak pathogen (Xanthomonas oryzae pv. Oryzicola), soybean bacterial macula pathogen (Xanthomonas anopolis pv. Glycines) and Xanthomonas campestris (Xanthomonas campestris) (see table 3 and figure 7); has weak inhibiting effect on tomato bacterial leaf spot germ (Pseudomonas syringae pv. Tomato).
TABLE 3 inhibitory Effect of fermentation filtrates of Streptomyces St-79 strains on 4 representative plant pathogenic bacteria
Note: different lower case letters indicate a significant difference at the P <0.05 level
Example 3 prevention Effect study of Streptomyces St-79 Strain fermentation filtrate on bacterial leaf blight of Rice
1 pathogen and Rice variety
(1) Actinomycetes for controlling effects: s. thioluteus (Streptomyces thioluteus) St-79 strain.
(2) Pathogenic bacteria: paddy rice bacterial blight (Xanthomonas oryzae pv. Oryzae, xoo)
(3) Rice variety: selecting Yongyou 1540 variety susceptible to rice bacterial leaf blight.
2 culture Medium
(1) Gao's No. one liquid medium: soluble starch 20g, KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 O0.01 g, water 1000mL, pH 7.4 (solid medium plus agar 20 g), used for the culture fermentation filtrate preparation of St-79 strain.
(2) NA solid medium: 3g of beef extract, 5g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH 7.2 (no agar is added in a liquid culture medium). Used for culturing Xoo pathogen.
3 Experimental methods
3.1 cultivation of Rice
Sterilizing plump rice seeds with uniform size in 75% alcohol for 15min, washing with sterile water for more than 5 times, placing the seeds on wet paper towel, placing in 25 deg.C incubator until the seeds germinate, sowing the germinated seeds in soil, and placing in incubator until the length of rice leaf exceeds 20 cm. The culture conditions are as follows: the light is 16h, the temperature is 30 ℃, the dark is 8h, the temperature is 25 ℃, the humidity is 65%, water is poured once every two days, and nutrient solution is sprayed once a week.
3.2 Culture of Xoo pathogen
Inoculating Xoo bacterium stored at 4 deg.C on NA medium plateActivating and culturing at 28 deg.C for 3d; single colonies were picked and inoculated into a 10mL tube with 3mL NA liquid base and activated to OD 600 =0.6; adding 1mL of bacterial liquid into 100mL of NA liquid culture medium, and culturing to OD 600 =0.6; centrifuging Xoo bacteria NA culture solution at 12000r/min for 5min, removing supernatant, adding sterile water, and making into OD 600 Xoo bacteria in water suspension of =0.6 for use in a control effect test.
3.3 Preparation of fermentation filtrate of St-79 Strain
St-79 strain 1 cake (diameter 5 mm) was taken with a punch, inoculated into Gao's No. I culture medium, and shake-cultured at constant temperature of 28 ℃ for 72 hours in a liquid volume of 50/250mL, initial pH 7.3, 160r/min, as a seed solution. Inoculating the seed solution into a Gao's No. I liquid culture medium according to the inoculation amount of 4%, and performing shake fermentation culture for 6d to obtain St-79 strain fermentation liquor. Centrifuging the mixture in a 50mL centrifuge tube at 12000r/min for 10min, filtering the supernatant with a 0.22 μm filter membrane, and removing residual spores to obtain fermentation filtrate. Diluting the fermentation filtrate with sterile water to 4 times, 5 times and 6 times of the stock solution for later use.
3.4 preliminary study of potted plant control
(1) Sterile Water blank control group (CK) 1 ): cutting leaves with aseptic scissors at 45 degrees, keeping the inoculated part wet all the time, and not performing other treatments;
(2) Xoo treatment Control (CK) 2 ): OD dipping with sterile scissors 600 Cutting leaves after 0.6 Xoo pathogen is suspended in clear water, and keeping the inoculated part moist all the time without other treatment;
(3) Treatment 1 (T) 1 ): OD dipping with sterile scissors 600 After leaf cutting and inoculation of a fresh water suspension of Xoo bacteria of which the concentration is not less than 0.6 for 6 hours, spraying St-79 strain fermentation filtrate diluted by 4 times (the fermentation filtrate is sprayed until liquid drips from leaves and is sprayed for 3 times, the interval is 1 hour each time, the same is carried out below), and performing moisture preservation measures on the inoculated parts;
(4) Treatment 2 (T) 2 ) OD dipping with sterile scissors 600 After leaf cutting and inoculation of 0.6 Xoo pathogen clear water suspension for 6 hours, spraying and diluting St-79 strain fermentation filtrate by 5 times, and making moisture preservation measures for the inoculated parts;
(5) Treatment 3 (T) 3 ) OD dipping with sterile scissors 600 Xo of =0.6After the leaves of the bacterial clean water suspension are cut and inoculated for 6 hours, st-79 strain fermentation filtrate diluted by 6 times is sprayed, and moisturizing measures are taken for the inoculated parts;
(6) Treatment 4 (T) 4 ): immediately spraying St-79 strain fermentation filtrate diluted 4 times after cutting leaves with sterile scissors at 45 degrees, inoculating OD with sterile cotton stick after 6h 600 =0.6 Xoo pathogen in clear water suspension to the leaf wound, and moisture preservation measures are taken for the joint part.
The above groups were all moisturized for 12h after treatment, 6 plants each, 5 leaves per plant (orthotopic leaves), 3 times per treatment.
And recording and counting the morbidity after the disease development tends to be stable. The disease condition is investigated by taking leaves as a unit, and the disease condition of the rice bacterial leaf blight is classified into 0-9 grades according to the following standard:
level 0: no disease spot is generated at the cut;
stage 1: the lesion area is less than 10% of the leaf area;
and 3, level: the lesion area is 11 to 25 percent of the leaf area;
and 5, stage: the lesion area is 26 to 45 percent of the leaf area;
and 7, stage: the lesion spot area is 46 to 65 percent of the leaf area;
and 9, stage: the lesion area is more than 65% of the leaf area.
According to the severity of the disease, calculating the disease index and the prevention and treatment effect according to the following formula:
disease index = ∑ (number of diseased leaves at each stage × corresponding stage value)/(investigation total number of leaves × highest-stage representative value) × 100
Relative control effect (%) = [ (control area disease index-treatment area disease index)/control area disease index ] × 100
Lesion suppression rate = (CK) 2 Lesion length-length of lesion treated)/CK 2 Length of lesion is 100 times
4 results of the experiment
The results of the experiment are shown in table 4 (fig. 8): the disease index of the rice bacterial leaf blight can be obviously reduced by processing the St-79 strain fermentation filtrate. Treatment 1 (T) 1 ) The relative prevention effect on the bacterial leaf blight of the rice is 82.49 percent, and the inhibition rate of the scab is 84.92 percent; treatment 2 (T) 2 ) For rice bacterial leaf blightThe relative prevention effect of the disease is 73.94 percent, and the inhibition rate of the scab is 78.75 percent; treatment 3 (T) 3 ) The relative control effect on the bacterial leaf blight of the rice is 67.32 percent, and the inhibition rate of the scab is 72.54 percent; treatment 4 (T) 4 ) The relative prevention effect on the bacterial leaf blight of the rice is 88.32 percent, the inhibition rate of the disease spots is 96.23 percent, and the control effect is better. From Process 1 (T) 1 ) And Process 2 (T) 2 ) And Process 3 (T) 3 ) The control effect result shows that the higher the concentration of the fermentation filtrate of the St-79 strain is, the better the control effect is. The experimental result also shows that the prevention and treatment effect of the first spraying of the St-79 strain fermentation filtrate is superior to that of the first inoculating of Xoo bacteria and then spraying of the fermentation filtrate, namely the prevention and treatment effect of the rice bacterial leaf blight is superior to that of the treatment effect.
TABLE 4 relative prevention of bacterial leaf blight of rice by fermentation filtrate of St-79 strain of Streptomyces
Note: different lower case letters indicate significant differences at the P <0.05 level
Example 4 Effect of fermentation filtrate of Streptomyces St-79 Strain on Rice seed Germination
1 rice variety: yongyou 1540 variety.
2 culture Medium
Gao's No. one liquid medium: soluble starch 20g, KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 O0.01 g, water 1000mL, pH 7.4 (solid medium plus agar 20 g), for culture of St-79 strain and fermentation filtrate preparation.
3 Experimental methods
3.1 Preparation of fermentation filtrate of St-79 Strain
St-79 strain 1 cake (diameter 5 mm) was taken with a punch, inoculated into Gao's first culture medium, and shake-cultured at constant temperature of 28 ℃ for 72 hours in a liquid volume of 50/250mL, initial pH 7.3, and initial pH 160r/min to serve as seed liquid. Inoculating the seed solution into a Gao's No. I liquid culture medium according to the inoculation amount of 4%, and performing shake fermentation culture for 6d to obtain St-79 strain fermentation liquor. Centrifuging the mixture in a 50mL centrifuge tube at 12000r/min for 10min, filtering the supernatant with a 0.22 μm filter membrane, and removing residual spores to obtain fermentation filtrate.
Diluting the fermentation filtrate with sterile water by 10 times (1/10), 50 times (1/50), 100 times (1/100), 500 times (1/500) and placing in a refrigerator at 4 deg.C for use.
3.2 Rice seed Germination treatment
Plump rice seeds are selected and soaked in sterile water at the temperature of 28 ℃ for pregermination for 24 hours.
3.3 influence of the fermentation filtrate seed soaking treatment on Rice seed Germination
Soaking the rice seeds after germination acceleration with 10 times (1/10), 50 times (1/50), 100 times (1/100) and 500 times (1/500) solutions diluted by fermentation filtrate stock solution and sterile water thereof respectively; the culture medium Gao's No. I was diluted by the above-mentioned multiple with sterile water as a Control (CK) 1 ) (ii) a Sterile water treatment as a common Control (CK) 2 ) Each treatment was repeated 3 times, each time 50 seeds. And (3) putting the seeds into a culture dish, respectively adding 20mL of each treatment solution into the culture dish, replacing the treatment solution for 1 time every 2d, observing the germination condition of the seeds every day from the seed soaking date, and taking the white exposed seeds as a germination standard.
Seed germination rate = (number of rice germinated seeds/total number of test rice seeds) × 100.
4 results of the experiment
The results of experiments on the influence of the fermentation filtrate of the Streptomyces St-79 strain on the germination rate of rice seeds are shown in Table 5 (FIG. 9), and neither the fermentation filtrate stock solution nor the culture medium stock solution treatment group has seed germination. Among all dilution multiples, the germination rate of the seeds treated by 1/100 times of fermentation filtrate is the highest and is obviously higher than that of the seeds treated by 1/100 times of Gao's I culture solution (P is less than 0.05); the germination rate of the rice seeds soaked in the fermentation filtrate diluted by sterile water is higher than that of the rice seeds soaked in the Gao's No. one culture solution with the same dilution times; the germination rates of the rice seeds soaked by 1/50 times, 1/100 times and 1/500 times of fermentation filtrate are obviously higher than those of the seeds soaked in sterile water (P is less than 0.05).
TABLE 5 influence of fermentation filtrate of Streptomyces St-79 strain on germination rate of rice seeds (%)
Note: different lower case letters indicate significant differences at the P <0.05 level
Description of the invention: the inhibition experiment of plant pathogenic bacteria was carried out on strain No. 12 (Zhanjun et al 2014) according to the experimental method described in example 2, and the results obtained were: the inhibition zones of Xanthomonas campestris and soybean bacterial macula is less than 10mm. The inhibition experiment effect of the plant pathogenic bacteria of CGMCC No.2725 is even inferior to that of the No. 12 strain.
The germination rate of strain fermentation filtrate of strain No. 12 (Zhanjun et al 2014) on rice seeds was tested according to the test method described in example 4, and the results were as follows: treating for 10 days, 1/50 fermenting filtrate, 1/100 fermenting filtrate, 1/500 fermenting filtrate and H 2 There was no significant difference in O.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by the person skilled in the art from the present disclosure are to be considered within the scope of the present invention.
Sequence listing
<110> Zhejiang university
<120> S.thioluteus St-79 and uses thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
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ccttcggggt ggattagtgg cgaacgggtg agtaacacgt gggcaatctg ccctgcactc 60
tgggacaagc cctggaaacg gggtctaata ccggatatga ccttcgagcg catgcttgaa 120
ggtggaaagc tccggcggtg caggatgagc ccgcggccta tcagcttgtt ggtggggtga 180
tggcctacca aggcgacgac gggtagccgg cctgagaggg cgaccggcca cactgggact 240
gagacacggc ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgaaa 300
gcctgatgca gcgacgccgc gtgagggatg acggccttcg ggttgtaaac ctctttcagc 360
agggaagaag cgaaagtgac ggtacctgca gaagaagcgc cggctaacta cgtgccagca 420
gccgcggtaa tacgtagggc gcaagcgttg tccggaatta ttgggcgtaa agagctcgta 480
ggcggcttgt cgcgtcggat gtgaaagccc ggggcttaac cccgggtctg cattcgatac 540
gggcaggcta gagttcggta ggggagatcg gaattcctgg tgtagcggtg aaatgcgcag 600
atatcaggag gaacaccggt ggcgaaggcg gatctctggg ccgatactga cgctgaggag 660
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgttggg 720
aactaggtgt gggcgacatt ccacgtcgtc cgtgccgcag ctaacgcatt aagttccccg 780
cctggggagt acggccgcaa ggctaaaact caaaggaatt gacgggggcc cgcacaagca 840
gcggagcatg tggcttaatt cgacgcaacg cgaagaacct taccaaggct tgacatacac 900
cggaaacggc cagagatggt cgcccccttg tggtcggtgt acaggtggtg catggctgtc 960
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgtcctgt 1020
gttgccagca tgcccttcgg ggtgatgggg actcacagga gactgccggg gtcaactcgg 1080
aggaaggtgg ggacgacgtc aagtcatcat gccccttatg tcttgggctg cacacgtgct 1140
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ctcagttcgg attggggtct gcaactcgac cccatgaagt tggagttgct agtaatcgca 1260
gatcagcatt gctgcggtga atacgttccc gggccttgta cacaccgccc gtcacgtcac 1320
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Claims (6)
1. S. thioluteus (Streptomyces thioluteus) St-79, characterized by: the preservation number is CCTCC NO: m2020962.
2. Use of the strain Streptomyces thioluteus (St-79) according to claim 1, characterized in that: promoting the germination of rice seeds.
3. Use according to claim 2, characterized in that: preventing and treating rice bacterial leaf blight disease.
4. Use according to claim 2, characterized in that: and (3) promoting the germination of rice seeds by using a diluent of the strain fermentation filtrate.
5. Use according to any one of claims 2 to 4, characterized in that: inhibiting plant pathogenic bacteria.
6. Use according to claim 5, characterized in that: the plant pathogenic bacteria are: rice bacterial leaf streak germ (Xanthomonas oryzae pv. Oryzae), soybean bacterial leaf streak (Xanthomonas axonopodis pv. Glycines) and Xanthomonas campestris (Xanthomonas campestris pv. Campholris).
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