CN115216430A - Streptomyces biocontrol strain and application thereof - Google Patents
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Abstract
The invention belongs to the field of biotechnology microorganisms, and particularly relates to streptomyces for biological control of rice blastStreptomycessp.QJK-30). The strain is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2019376. Experiments show that: streptomyces (I), (II)Streptomycessp.QJK-30 CCTCC NO: m2019376) fermentation liquor has good biological control effect on rice blast, and the relative control effect is 70.9%.
Description
Technical Field
The invention belongs to the field of biotechnology microorganisms, and particularly relates to streptomyces (I) for efficiently preventing and treating rice blastStreptomyces sp.QJK-30) biocontrol strain and application thereof.
Background
The rice blast is a destructive rice fungal disease, the yield loss of rice is 10% -30% every year, the yield loss can reach 50% in severe cases, even the rice blast is extremely low, and the rice lost due to the rice blast is enough to live 6000 million people every year all over the world. The rice blast is easy to occur at the 3-4 leaf stage, the tillering stage and the heading stage of rice. The pathogen is Pyricularia oryzae (Magnaporthe oryzae) The physiological factors are various and have variable heights, and the influence of geography and climate is large, so that the treatment difficulty is large.
At present, the method for preventing and controlling rice blast mainly comprises the following steps: breeding and utilizing disease-resistant variety, agricultural cultivation management and chemical pesticide application. The breeding difficulty of the disease-resistant variety is large, and the period is long. In the traditional agriculture, a large amount of manpower and material resources are needed for preventing and controlling rice blast in cultivation measures, and in order to pursue high yield of rice, high-yield and multi-tiller improved seeds and cultivation technologies of seedling throwing, high density and high nitrogen fertilizer application are adopted, so that a proper growth environment is created for pathogenic bacteria, and the prevention and control of the agricultural cultivation measures are difficult and serious. Chemical pesticides have high residue, are harmful to people and livestock, pollute the environment, have environmental risk, and cause drug resistance after long-term use. The antagonistic microorganisms are utilized to prevent and control plant diseases, and the green prevention and control of the plant diseases are more and more emphasized due to the characteristics of good selectivity, easy degradation, environmental friendliness and the like. Research shows that the rhizosphere beneficial microorganisms have good affinity to plants, can easily survive and colonize on the body surface or in vivo of the plants after being applied, and have durable and stable prevention and treatment effects. Although some antagonistic strains or potential biocontrol strains against Pyricularia oryzae have been found, e.g., pseudomonas (Pseudomonas oryzae)Pseudomonas sp.) Bacillus bacteria (b), (b)Bacillus sp.) And certain Streptomyces (I), (II)Streptomyces sp.) However, different antagonistic microbes have different mechanisms of action and methods of use. Different production applications require different biocontrol strains, so that more effective biocontrol bacteria are screened to be necessary.
Disclosure of Invention
The invention aims to provide streptomyces (Streptomyces)Streptomyces sp.QJK-30) can be used as a biological pesticide to prevent and control rice blast.
Streptomyces (I) of the present inventionStreptomycessp.QJK-30) is obtained by adopting a soil continuous gradient dilution method in the rice rhizosphere soil and combining an indoor confrontation test for separation and screening. The streptomyces is preserved in China center for type culture Collection (Wuhan university), the preservation unit is called CCTCC for short, and the preservation number is CCTCC NO: m2019376, the preservation date is 5 months and 20 days in 2019. It has the following microbiological properties: gram-positive bacteria, compact spiral spore chain, liquefied gelatin, peptone positive, catalase positive, oxidase positive, esterase positive, starch hydrolysis positive, cellulose hydrolysis negative, and no H production 2 S; can utilize carbon sources such as D-glucose, D-fructose, sucrose, melibiose, D-xylose, D-raffinose, L-rhamnose, etc., and cannot utilize glycerol, inositol, L-arabinose, D-mannitol, D-galactose and alpha-lactose; the nitrogen source is widely available (except that D-serine and L-arginine are not available). After 7 days of culture on the NA culture medium, the bacterial colony is smooth, the mycelium in the medium is olive-brown, and the aerial mycelium is rich and white and has no soluble pigment. And (3) culturing in an NB liquid culture medium for 3 days, wherein the fermentation liquor is golden yellow, clear and transparent, the thalli grow vigorously, and the bacterial balls are fine.
Streptomyces (I) of the present inventionStreptomyces sp.QJK-30), conditions for detecting viability: (1) Nutrient Agar (NA) culture medium, 10g of peptone, 5 g of sodium chloride, 5 g of beef extract, 15 g of agar powder and 1L of distilled water, wherein the pH value is 7.2-7.4, and the culture is carried out at 28 ℃. The laboratory liquid culture uses Nutrient Broth (NB) medium. (2) the mass fermentation medium is a millet medium: 10g of glucose, 3 g of peptone and 2.5 g of 8194NaCl and CaCO 3 81942 g, millet 10g, distilled water 1000 mL, pH 7.2-7.4 8194g.
The microbial preparation can be used as a biological pesticide for preventing and treating rice blast, and the prevention and treatment effect can reach 70.9%.
The invention has the beneficial effects that:
(1) The streptomycete QJK-30 is a beneficial actinomycete of plant rhizosphere, can be colonized on the surface and in the body of a plant, and has lasting and stable prevention and treatment effects.
(2) The streptomycete QJK-30 is environment-friendly.
(3) The streptomycete QJK-30 has obvious control effect on rice blast.
(4) The streptomycete QJK-30 has the advantages of simple culture condition, easy storage, easy industrial production and good development and application potential.
Description of the drawings:
FIG. 1 Streptomyces (I) according to the example of the present inventionStreptomyces sp.QJK-30) single colony morphology on Gao's first medium;
FIG. 2 Streptomyces (I), (II) according to the example of the present inventionStreptomyces sp.QJK-30) (based on 16S rDNA sequences);
FIG. 3 Streptomyces (I), (II) according to the example of the present inventionStreptomyces sp.QJK-30) has inhibitory effect on the growth of rice blast fungus hypha.
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to the following examples and drawings.
Example 1: separating and screening streptomycete QJK-30.
The streptomyces of the invention has the generic name ofStreptomycesThe seed name isStreptomycessp. the plant name is QJK-30, which is separated from the rice rhizosphere soil of the rice planting area in the Mongolian area of Mongolian monsoon of the Mongolian city of Hunan province and obtained by screening through a soil continuous gradient dilution method and a flat plate opposite experiment, and the screening method belongs to the conventional experimental method in the field.
The specific method and process are described as follows: preparing rhizosphere soil sample (about 10 cm deep from soil surface) 1 g into 10 mL sterile water -2 Shaking the soil suspension at 28 ℃ and 180 rpm for 30 min, standing for 30 min, taking a supernatant, continuously and gradiently diluting, coating the supernatant on a Gao's first culture medium plate, and culturing at 28 ℃. And (3) selecting a single colony, and carrying out streak culture on a Gao's first culture medium plate to obtain a streptomycete strain with the number of QJK-30.
The formula of the Gao's No. one culture medium is as follows: 20 g soluble starch, 1 g KNO 3 ，0.5 g NaCl，0.5 g K 2 HPO 4 ·3H 2 O，0.5 g MgSO 4 ·7H 2 O，0.01 g FeSO 4 ·7H 2 O,16 g agar, 1000 mL distilled water, pH 7.4-7.6.
Example 2: streptomyces (I), (II)Streptomycessp, QJK-30) of a test piece
1. Morphological identification
After the QJK-30 strain is cultured on a Gao's No. I culture medium for 7 days at a constant temperature of 28 ℃, the colony is circular, the texture is compact, and the aerial hyphae are white, as shown in figure 1.
2. Physiological and biochemical identification
The QJK-30 strain is positive in gram stain, oxidase, catalase, esterase, urease and nitrate reduction tests; negative methyl Red test, V-P test and indole production test, and no H production 2 S; gelatin liquefaction, milk peptonization, casein hydrolysis, starch hydrolysis, utilization of citrate, and no hydrolysis of cellulose, as shown in table 1.
TABLE 1 physio-biochemical characteristics of Streptomyces sp. QJK-30
| Gram stain | + | Decomposition of cellulose | - |
| Sugar fermentation ability | Does not produce acid | Nitrate reduction | + |
| Liquefaction of gelatin | + | Oxidase enzyme | + |
| Milk peptone | + | Contact enzyme | + |
| Starch hydrolysis | + | MR test | - |
| Esterase | + | V-P test | - |
| Indole production | - | H 2 S generation | - |
| Citrate utilization | + | Urease | + |
| Hydrolysis of casein | + |
3. Molecular identification
Taking single colony of the QJK-30 strain, performing shake culture for 5d (28 ℃,150 rpm) in a Gao's I liquid culture medium, centrifuging at 12000 rpm for 1min, removing supernatant, and collecting the thallus. The operation was carried out by using a bacterial genome DNA extraction kit (Biotechnology engineering (Shanghai) Co., ltd.) according to the instructions. PCR amplification was carried out using the genomic DNA of the QJK-30 strain as a template, based on the prokaryotic microorganism 16S rDNA universal primers 27F (5-. 100.μ l PCR reaction: 10 μ l of 10 XPCR buffer (containing MgCl) 2 ) 2. Mu.l of 10 mmol/L dNTP, 5. Mu.l of 10. Mu. Mol/L27F primer, 5. Mu.l of 10. Mu. Mol/L1492R primer, 5. Mu.l template DNA (200 ng), 2.5U Taq polymerase, and after double distilled water was added to the final volume and mixed well, amplification was performed on a PCR instrument: after denaturation at 95 ℃ for 4 min, PCR cycles are carried out, wherein each cycle is 94 ℃ 30s,50 ℃ 30s and 72 ℃ for 1min, and 30 cycles are total; then extended for 7min at 72 ℃. The PCR amplification results were analyzed by detection on a 1% agarose gel electrophoresis (1% is the mass percentage of agarose). The PCR product is recovered by using a recovery kit and sent to biological engineering (Shanghai) company Limited for sequence determination. The sequencing result is spliced to obtain the 16S rRNA gene sequence of the QJK-30 strain, the length is 1402bp, and the accession number of NCBI (https:// www.ncbi.nlm.nih.gov /) is MK791670.
To be provided withMycobacterium. tuberculosisThe 16S rRNA sequence of the strain is an outer group, a phylogenetic tree is constructed, and the result shows that the QJK-30 strain and the model strainStreptomyces albospinus NBRC 13848(T)、Streptomyces kasugaensis BCRC 12349(T)、Streptomyces cellulofavus NRRC B-2493 (T) is positioned under the same small branch, and is combined with the thallus morphology, the colony characteristics and the physiological and biochemical characteristics to be preliminarily identified as streptomyces.
Example 3: an in vitro bacteriostatic activity determination experiment of streptomyces QJK-30 on rice blast germs.
The rice blast R01-1 strain (preserved in the laboratory of the applicant) is used as a test strain, and the strain is measured by a plate confronting method. Reactivating the QJK-30 strain and the rice blast fungus R01-1 strain, punching by using a sterile puncher (7 mm), clamping a rice blast fungus R01-1 agar block by using sterile tweezers, placing the rice blast fungus R01-1 agar block in the center of a PDA (personal digital assistant) plate, placing the QJK-30 strain agar block at a position 3 cm away from the center of the PDA plate, using the inoculated sterile agar block as a control, culturing at 28 ℃ until the pathogenic fungi grow over the whole plate, observing the bacteriostatic effect and calculating the bacteriostatic rate.
Bacteriostasis rate (%) = (Rp-Rt)/Rp multiplied by 100%
Wherein Rp represents the hyphal growth radius of the control group, rt represents the hyphal growth radius (close to the antagonistic bacteria side) of the treatment group, and the hyphal growth radius is calculated by subtracting the radius (3.5 mm) of the fungus cake from the measured average value.
The PDA culture medium comprises the following components: 200 Potato, 20 g glucose, 16 g agar, 1000 mL distilled water, natural pH.
As shown in FIG. 3, streptomyces QJK-30 has a significant inhibitory effect on the hypha growth of rice blast fungus R01-1 strain, with an inhibition rate of 78.8%.
Example 4: a field test of streptomycete QJK-30 for preventing and controlling rice blast.
The field test site is carried out in Jinchancun of Yueshan mountain town of Xiangxiang city, hunan province, and the site naturally generates rice blast all year round. The rice variety is hybrid Zhongdao Longyou Huazhan. Dividing the field block into 4 × 5 m small areas, performing random block test, spraying rice seedlings 20d and 40d after transplanting, treating by 4 treatments, repeating for 3 times: (1) clear water control, 750L/ha; (2) Streptomyces QJK-30 fermentation suspension (1X 10) 8 cfu/mL), 750L/ha; (3) Tricyclazole (75% wettable powder), application amount 300 g/ha, adding water 750L/ha. At 40d after the second spray treatment, the control effect (control effect) was calculated according to the rice blast test survey code (GB/T15790-1995). Disease index = Σ [ number of diseased leaves at each level x relative disease level value at each level]/[ survey of Total leaf number]X highest disease level value. Control effect (%) = (control disease index-treatment disease index)/control disease index x 100.
Test results show that the control effect of the streptomycete QJK-30 strain on rice blast reaches 70.9 percent, and the control effect is not obviously different from that of tricyclazole (72.4 percent) (P is more than 0.05) (Table 2).
TABLE 2 field control of Streptomyces QJK-30 against Pyricularia oryzae
| Treatment of | Index of disease condition | Control effect (%) |
| Control of | 13.4 | - |
| QJK-30 | 3.9 | 70.9 |
| Tricyclozole | 3.7 | 72.4 |
Claims (5)
1. A Streptomyces (I)Streptomyces sp.QJK-30), is preserved in China Center for Type Culture Collection (CCTCC) for short, and the preservation number is CCTCC NO: m2019376.
2. The Streptomyces of claim 1 (S.) (Streptomyces sp.QJK-30) biocontrol strain, characterized in that said Streptomyces (Streptomyces: (Streptomyces)Streptomyces sp.QJK-30) is obtained by adopting a soil continuous gradient dilution method in the rhizosphere soil of the rice and combining the indoor confrontation test for separation and screening.
3. The Streptomyces of claim 1 (i: (la), (lb), and (lb)Streptomyces sp.QJK-30) biocontrol strain, characterized in that said Streptomyces (Streptomyces: (Streptomyces)Streptomyces sp.QJK-30) have the following microbiological properties: gram-positive bacteria, compact spiral spore chain, liquefied gelatin, peptone positive, catalase positive, oxidase positive, esterase positive, starch hydrolysis positive, cellulose hydrolysis negative, and no H production 2 S; can utilize carbon sources such as D-glucose, D-fructose, sucrose, melibiose, D-xylose, D-raffinose, L-rhamnose, etc., and cannot utilize glycerol, inositol, L-arabinose, D-mannitol, D-galactose and alpha-lactose; the nitrogen source is widely utilized (except that D-serine and L-arginine are not utilized); after 7d of culture on the NA culture medium, the bacterial colony is smooth, the mycelium in the medium is olive-brown, and the aerial mycelium is rich and white and has no soluble pigment; and (3) culturing in an NB liquid culture medium for 3 days, wherein the fermentation liquor is golden yellow, clear and transparent, the thalli grow vigorously, and the bacterial balls are fine.
4. The Streptomyces of claim 1 (i: (la), (lb), and (lb)Streptomyces sp.QJK-30) biocontrol strain, characterized in that said streptomyces (Streptomyces)Streptomyces sp.QJK-30) and conditions for detecting viability: (1) Nutrient Agar (NA) culture medium, 10g of peptone, 5 g of sodium chloride, 5 g of beef extract, 15 g of agar powder and 1L of distilled water, wherein the pH value is 7.2-7.4, and the culture is carried out at 28 ℃; the laboratory liquid culture adopts Nutrient Broth (NB) culture medium; (2) the large-scale fermentation medium is a millet medium: 10g of glucose, 3 g of peptone, 2.5 g of NaCl, caCO 3 2 g, 10g of millet and 1000 mL of distilled water, and the pH value is 7.2-7.4.
5. The use of the biocontrol strain of streptomyces as defined in claim 1 in preventing and controlling rice blast.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002034884A1 (en) * | 2000-10-25 | 2002-05-02 | Green Biotech Co., Ltd. | Streptomyces kasugaensis inhibiting the fungal pathogens of plant |
| CN1519310A (en) * | 2003-05-22 | 2004-08-11 | 郑爱萍 | Strain of streptomycete for preventing sheath blight of rice |
| CN101984044A (en) * | 2010-01-19 | 2011-03-09 | 四川农业大学 | Streptomyces strain for preventing rice sheath disease and rice blast disease |
| CN109762753A (en) * | 2018-11-01 | 2019-05-17 | 福建省农业科学院植物保护研究所 | One plant of special habitats streptomycete bacterial strain and its application |
| CN110607249A (en) * | 2018-12-27 | 2019-12-24 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
| CN113005048A (en) * | 2020-12-14 | 2021-06-22 | 河南农业大学 | Streptomyces nigricans CYS22, metabolite thereof and application thereof |
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- 2022-08-01 CN CN202210918289.XA patent/CN115216430A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002034884A1 (en) * | 2000-10-25 | 2002-05-02 | Green Biotech Co., Ltd. | Streptomyces kasugaensis inhibiting the fungal pathogens of plant |
| CN1519310A (en) * | 2003-05-22 | 2004-08-11 | 郑爱萍 | Strain of streptomycete for preventing sheath blight of rice |
| CN101984044A (en) * | 2010-01-19 | 2011-03-09 | 四川农业大学 | Streptomyces strain for preventing rice sheath disease and rice blast disease |
| CN109762753A (en) * | 2018-11-01 | 2019-05-17 | 福建省农业科学院植物保护研究所 | One plant of special habitats streptomycete bacterial strain and its application |
| CN110607249A (en) * | 2018-12-27 | 2019-12-24 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
| CN113005048A (en) * | 2020-12-14 | 2021-06-22 | 河南农业大学 | Streptomyces nigricans CYS22, metabolite thereof and application thereof |
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