CN114456973A - Streptomyces rochei strain in tobacco and application thereof in prevention and control of tobacco diseases - Google Patents
Streptomyces rochei strain in tobacco and application thereof in prevention and control of tobacco diseases Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the field of agricultural microorganisms, and discloses a streptomyces roqueforti strain in tobacco and application thereof in prevention and control of tobacco diseases, wherein the preservation number of the strain is CCTCC NO: M20211629. The strain is reported for the first time to be simultaneously used for preventing and controlling tobacco black shank, nematode, anthracnose and brown spot, and has no cross drug resistance with the prior bactericide and insecticide. Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because the pesticide is from a natural environment, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and particularly relates to a streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases.
Background
Tobacco is an important economic crop in China, but soil-borne diseases are serious for a long time due to continuous cropping, wherein tobacco black shank and nematode are 2 important diseases.
The tobacco black shank is a root-stem disease caused by Phytophthora nicotianae (Phytophthora nicotiana), occurs in each big smoke area in China, causes hundreds of millions of yuan of loss every year, and is an important factor for restricting the high-quality and safe production of tobacco in China. The control measures of the tobacco black shank comprise agricultural control, disease-resistant variety, chemical control and biological control[The disease-resistant variety is difficult to cultivate and has strong regionality; the chemical pesticide is easy to cause the problems of pesticide residue, environmental pollution, drug resistance and the like after being used in large quantities for a long time. With the proposal of the green prevention and control concept, the environment-friendly high-efficiency antagonistic bacteria becomes an important measure for preventing and controlling the black shank of the tobacco.
In recent years, the Root-knot nematode (Root-knot nematode) disease of tobacco tends to be aggravated year by year in all tobacco regions in China, and the harm is very serious particularly in the tobacco main producing areas such as Henan, Yunnan, Shandong, Shanxi, Guangxi, Sichuan and the like. The tobacco root knot nematode disease is caused by plant root knot nematodes (Meloidogyne spp.), and mainly damages the root of tobacco, and seriously influences the healthy development of the tobacco. Meanwhile, mechanical wounds caused by root-knot nematode infection on tobacco are easy to induce various tobacco root diseases, such as tobacco black shank, fusarium root rot, root black rot, bacterial wilt and the like, and the formed composite infection diseases cause the growth vigor of tobacco plants to be weak and the resistance to be poor, so that the occurrence and the prevalence of various diseases of the tobacco are aggravated. The root-knot nematode infection can induce and aggravate the occurrence of other soil-borne diseases of tobacco, so that the development of the biocontrol preparation with double functions on the root-knot nematode and the soil-borne diseases of the tobacco has important significance.
Aiming at the problems, the invention separates out a new strain of Streptomyces rochei YC117, which has good control effect on tobacco black shank and root knot nematode and has better colonization ability on tobacco root system. At present, the endophytic Streptomyces rochei with bacteriostasis has been reported, for example, a fermentation liquor of a strain ZZ-9 (the growth promotion effect of a fermentation liquor of the Streptomyces rochei ZZ-9 strain on wheat seedlings) has certain insecticidal activity on Meloidogyne incognita, and the bacteriostasis rate of Fusarium oxysporum is 53.27%. The L-25 strain (biological prevention and control of soil-borne tobacco bacterial wilt and mechanism research thereof) separated from a tobacco field has good bacteriostatic activity on Laurella Ralstonia solanacearum and Fusarium oxysporum which are pathogenic bacteria of tobacco bacterial wilt, and the YC117 strain has no bacteriostatic activity on Laurella. Strains LN204, LN221 and LN 2473 isolated from tobacco rhizosphere (screening and identifying of tobacco rhizosphere in Luoyang tobacco zone against phytophthora nicotianae actinomycetes) have bacteriostatic activity of 100% for tobacco black shank pathogenic bacteria, and YC117 has bacteriostatic activity of 73.67% for tobacco black shank pathogenic bacteria. In addition, the prevention and control of soil-borne diseases are not only related to the bacteriostatic activity of strains and the colonization ability of root systems, but also related to the regulation and control effect of the biocontrol microbial inoculum on rhizosphere microbial community structures, the disease resistance of induced plants and the like, so different strains of the same type of biocontrol bacteria can have differences in the aspects of regulating and controlling the rhizosphere microbial community structures, inducing the disease resistance of the plants and the like.
Disclosure of Invention
The invention aims to provide a separated streptomyces, which is Streptomyces rochei YC117 with the preservation number of CCTCC NO: M20211629. The streptomyces can be used for preventing and controlling tobacco diseases such as tobacco black shank, brown spot, anthracnose, nematode disease and the like.
The invention also aims to provide the application of the streptomyces rochei YC117 in the preparation of plant pathogen inhibitor or nematode pesticide, in particular to the preparation of the drug for preventing and treating tobacco diseases.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
an isolated Streptomyces isolated from tobacco roots, identified as Streptomyces rochei, which has been deposited at 12/15 months 2021 in the chinese culture collection under the taxonomic nomenclature: streptomyces rochei YC 117; the preservation number is CCTCC NO of M20211629; a place: wuhan university in Wuhan China.
The strain grows well on an ISP2 culture medium, the color of aerial hyphae is white to off-white, the color of intrabasal hyphae is yellow, soluble pigment is yellow, spore filaments are straight or flexible, the spores are oval, the center of a colony is raised, and the edge is white and radial (figure 1).
The application of Streptomyces rochei YC117 in the preparation of a plant pathogenic bacteria inhibitor is as follows: tobacco Phytophthora (Phytophthora nicotiana), Pythium aphanidermatum (Pythium aphanidermatum), tobacco anthracnose (Colletotrichum nicotiana), Alternaria alternata (Alternaria alternata), Sclerotinia sclerotiorum (sclerotirotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), and/or Botrytis cinerea (Botrytis cinerea).
The invention also comprises the application of the Streptomyces rochei YC117 in the preparation of the nematode pesticide.
Compared with the prior art, the invention has the following advantages:
(1) the invention provides a streptomyces lavendulae strain which is reported for the first time to be used for simultaneously preventing and controlling tobacco black shank disease, brown spot disease, anthracnose and nematode disease. Has no cross resistance with the prior bactericide and insecticide.
(2) Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because of being from natural environment.
Drawings
FIG. 1 is a diagram showing the spore, hypha and colony morphology of Streptomyces rochei YC117 strain.
FIG. 2 is a schematic diagram of Streptomyces rochei YC117 solid fermentation microbial inoculum.
FIG. 3 shows the bacteriostatic spectrum of Streptomyces rochei YC 117.
FIG. 4 is a schematic diagram of the effect of Streptomyces rochei YC117 microbial inoculum on preventing and treating tobacco black shank.
FIG. 5 is a schematic diagram of the effect of Streptomyces rochei YC117 microbial inoculum on preventing and treating tobacco nematode disease.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
obtaining and identifying Streptomyces rochei YC117
A separated Streptomyces is separated from the root system of tobacco in En Xuan county of Enshi, belongs to Streptomyces rochei (table 1 and table 2) after being combined with physiological and biochemical identification through 16s rDNA, is delivered to the China center for type culture collection at 12 months and 15 days in 2021 for collection, and is classified and named: streptomyces rochei YC 117; the preservation number is CCTCC NO of M20211629; a place: china, wuhan university.
In the present invention, Streptomyces rochei YC117 or simply referred to as YC 117.
Morphological characteristics: the aerial hyphae are white to off-white, the basal hyphae are yellow, the soluble pigment is yellow, the spore silks are straight or flexible, the spores are oval, the center of the colony is convex, and the edge is white and radial.
TABLE 1 BLAST results of Gene sequencing of strain YC 11716S rRNA
TABLE 2 physiological and biochemical characteristics of Strain YC 117-carbon Source utilization
+: positive reaction; -: negative reaction; w-weakly positive reaction
Example 2:
fermentation of Streptomyces rochei YC 117:
preparation of Streptomyces rochei YC117 seed liquid: comprises activating slant-stored strain on ISP-2 flat plate, selecting and inoculating to 100 ml seed culture medium, and culturing on a shaking table at 28 deg.C and 160 rpm for 4 days; obtaining the seed liquid.
The formula of the seed culture medium is as follows: 1L of the culture medium comprises 20g of glucose, 8g of soybean peptone, 0.30g of potassium dihydrogen phosphate, 0.5g of calcium carbonate, 20 mu L of soybean oil and the balance of water.
A solid-state fermentation method of Streptomyces rochei YC117 comprises the following steps:
preparing liquid fermentation liquor: inoculating Streptomyces rochei YC117 seed liquid into fermentation culture medium at 30 deg.C and stirring speed of 500rpm/min, wherein the fermentation culture medium content is 60% of tank volume, and fermenting for 4 days.
The formula of the fermentation medium comprises: 3% of glucose, 3% of soybean peptone, 0.3% of yeast powder, 0.5% of calcium carbonate and 0.5% of sodium chloride; pH 7, and the balance water.
Sterilizing perlite at high temperature, and uniformly mixing the fermentation liquor and the perlite, wherein the mixing ratio is 4 mL: 1g of the perlite and the perlite are mixed, and the mixture is spread in a plastic box, the height of the perlite is 5cm, the fermentation temperature is 30 ℃, and the fermentation time is 14 d; drying at 100 ℃ and crushing for later use; the particle size of the perlite is as follows: 3mm-5 mm.
After fermentation with perlite substrate, the spore number of Streptomyces rochei YC117 is 2.5 × 1010Spores per gram (FIG. 2), isolated and streaked, free of undesired bacteria, fermented uniformly and completely, for use in the following examples;
example 3:
bacteriostatic spectrum of Streptomyces rochei YC117
The tobacco black shank pathogenic bacteria are separated from tobacco black shank plants, Pythium aphanidermatum (Pythium aphanidermatum) is separated from cucumber damping-off plants, and 2 kinds of pathogenic bacteria are stored on a V8 inclined plane at 10 ℃.
The tobacco anthrax (Colletotrichum nicotianae) and Alternaria alternata (Alternaria alternata) were isolated from tobacco anthracnose and Alternaria alternate leaves, the Fusarium oxysporum (Fusarium oxysporum) was isolated from a tobacco-diseased strain, the Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) was isolated from a production-diseased strain, the Rhizoctonia solani (Rhizoctonia solani) was isolated from rice sheath blight leaves, and the Botrytis cinerea (Botrytis cinerea) was isolated from strawberry-diseased fruits. Laurella (Ralstonia solanacearum) was isolated from a strain of tobacco bacterial wilt and stored in a glycerin tube at-20 ℃. Transferring the slant fungus strain into PDA plate for activation, and culturing at 30 deg.C for 6 d. And (3) placing YC117 bacterial discs at two ends of a PDA flat plate, inoculating the pathogenic bacterial discs with a 4mm puncher after 24h, taking the inoculated culture medium bacterial discs as a blank control, and keeping the distance between the pathogenic bacteria and the YC117 bacterial discs to be 25 mm. And after the contrast grows over the flat plate, counting the diameters of the treated and contrasted bacterial colonies, and calculating the bacteriostasis rate.
The inhibition rate is [ (control colony diameter-treated colony diameter)/(control colony diameter-cake diameter) ] × 100%
After the laonella is obtained by drawing lines from a glycerol tube, the laonella is transferred into an NB liquid culture medium shaker for shaking culture at the temperature of 37 ℃ at 180r/min for 48 hours for later use. Melting the NA culture medium, pouring the medium into a flat plate at about 45 ℃, then adding the Laurella bacterial liquid, wherein the ratio of NA to bacterial liquid is 50:1, and fully and uniformly mixing. And then punching the bacteria-carrying plate by using a sterilized Oxford cup, adding 200 mu L YC117 fermentation liquor into the holes, and culturing at 37 ℃ for 48h to observe whether a bacteriostatic circle is generated. The result shows that Streptomyces rochei YC117 has no bacteriostatic activity on Laurella.
In vitro bacteriostasis experiments show that YC117 has good bacteriostasis activity to pathogenic bacteria such as phytophthora nicotianae, anthrax of tobacco, alternaria alternata and the like (Table 3, figure 3).
TABLE 3 bacteriostatic Activity of YC117 against pathogenic bacteria
Example 4: streptomyces rochei YC117 for preventing and treating tobacco black shank
Directly sowing tobacco seeds in 32-hole plug trays, conventionally culturing in a greenhouse, transplanting in a seedling pot after 15 days, and using for a pot experiment after 30 days. Activating Phytophthora nicotianae from inclined plane to PDA plate, propagating on V8 plate, culturing at 28 deg.C for 12 days, brushing spores, placing in 4 deg.C refrigerator for half an hour, adjusting spores to 10 with blood counting plate5Spores/ml. YC1175 microbial inoculum is diluted by 100 times and irrigated with roots (1 gram is added with 100L of water), the root irrigation amount is 20ml per plant, and phytophthora nicotianae spores are inoculated after 24 hours, and the root irrigation amount is 3ml per plant. The treatment is repeated for 3 times, 15 seedlings are repeated for each time, clear water is used as a reference, the reference and the treatment disease index are counted after 15 days, and the disease incidence is calculated.
The disease index grading standard of the tobacco black shank refers to the tobacco industry standard of the people's republic of China (YC/T39-1996)
Disease index ∑ (number of plants at each stage × rank)/(total number of investigated plants × highest representative rank) × 100.
The prevention and treatment effect is (contrast disease index-treatment disease index)/contrast disease index multiplied by 100 percent
The result of the potted plant growth shows that the prevention effect of the YC1175 microbial inoculum diluted by 100 times on the tobacco black shank is 100 percent (figure 4).
Example 5: streptomyces rochei YC117 for preventing and treating tobacco nematode disease
The test is carried out in En, Schoen, Hubei province, the root YC117 microbial inoculum is irrigated by 200 times after the tobacco is transplanted and fixedly planted for 10 days, the root is irrigated once every 10 days, 200 mL/plant, clear water is irrigated in contrast, each treatment is repeated for 3 times, and each treatment is repeated for 40m2And irrigating roots for 3 times in the test, counting root knot indexes of each treatment before harvesting, and calculating the control effect.
The occurrence condition of tobacco diseases is investigated according to the GB/23222-2008 tobacco disease and insect pest grading and investigation method.
The root knot index ∑ (number of plants at each stage × rank)/(total number of investigated plants × highest representative rank) × 100.
The control effect is (contrast root knot index-treatment root knot index)/contrast root knot index multiplied by 100 percent
The test result shows that the YC117 microbial inoculum has good prevention and control effect on the tobacco root-knot nematode, and the prevention effect reaches 62.98 percent (shown in a table 4 and a figure 5)
TABLE 4 YC117 fungicide field control of tobacco root-knot nematodes
| Treatment of | Root knot index | Control effect (%) |
| YC117 | 26.78 | 62.98 |
| Control | 72.34 | - |
Example 6: streptomyces rochei YC117 has the function of colonization of tobacco roots
YC117 microbial inoculum was made into 0.1 hundred million cfu/mL suspension with clear water, and the control agent was 2 hundred million cfu/g streptomyces rochei powder (T-203 strain, trade name:
) Will beIt was made into a suspension of 0.1 hundred million cfu/mL with clear water. 2 kinds of Streptomyces rochei bacterial suspension are irrigated in a seedling culture pot for growing 40d tobacco, 20 ml/plant, each treatment is repeated for 3 times, and each treatment is repeated for 10 plants. After 15 days, randomly digging 5 plants from each repeated pot, cutting off root systems, washing the root systems clean by using tap water, and then sucking water on the surface of the roots by using absorbent paper. Cutting off fibril, weighing 1.00g fibril sample on a balance, bundling into small bundles, soaking in 75% (V/V) ethanol for 1-2s, removing bubbles on the surface of the fibril, and adding water to the solution with concentration of 1 g.L-1Soaking in mercuric chloride solution for 2min, taking out, and washing with sterile water for 5 times. And (3) putting the mixture into a sterile mortar added with a small spoon of sterilized quartz sand and 10mL of sterile water for full grinding, so that the streptomyces colonized in roots is released. After proper dilution, spreading the mixture on a Gao's No. 1 plate for culture at 28 ℃, and adding sterilized K before pouring the Gao's No. 1 culture medium into a dish2Cr2O7Making K in the culture medium2Cr2O7The final mass concentration of (3) is 80 mg/L). Counting after morphological comparison and identification are carried out on the streptomycete strains on the synchronous culture flat plate, and calculating the number of the colonized streptomycete viable bacteria (colonizing density) on each gram of roots.
The test result shows that the YC117 strain has good colonization ability in tobacco root system, and the colonization density in the tobacco root system after 15 days is 2.3 multiplied by 105cfu/g, whereas the T-203 strain has no colonization ability in tobacco.
Example 7: effect of Streptomyces rochei YC117 on tobacco salicylic acid content
In the 6-leaf stage of healthy tobacco leaves, diluting YC117 microbial inoculum by 100 times, irrigating roots by 20 mL/plant, irrigating clear water in contrast, repeating treatment for 3 times, repeating seedling for 5 plants, extracting salicylic acid according to the specification of a salicylic acid content test box (SaA-4-Q salicylic acid content test box) after 48h, detecting salicylic acid content at 240nm by using a high performance liquid chromatograph, preparing a standard curve by using a standard product with known concentration, and obtaining a linear relation y of 30.056x +0.915(R is R) according to the peak area and the concentration20.9999) was calculated. Salicylic acid is currently considered to be the main signal molecule for acquiring resistance of a plant production system, and the acquisition of resistance of the salicylic acid-mediated system is already carried out in transgenic plants of model plants such as arabidopsis, tobacco, cucumber and the likeAnd mutant plants were confirmed in a series of scientific experiments. After YC 11748 h is sprayed, the salicylic acid content in the tobacco leaves is 7.41 mug/g, the contrast is 4.91 mug/g, which indicates that YC117 has the effect of inducing resistance to the tobacco.
Claims (3)
1. An isolated Streptomyces rochei (S. (L.))Streptomyces rochei) The preservation number is CCTCC NO: M20211629.
2. The use of Streptomyces rochei as claimed in claim 1 in the preparation of a plant pathogen inhibitor, said pathogen being Phytophthora nicotianae (Phytophthora nicotianae)Phytophthora nicotianae) Pythium aphanidermatum (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B) and a)Pythium aphanidermatum) Tobacco anthracnose bacterium (Colletotrichum nicotianae) Alternaria alternata (Alternaria alternata) Sclerotinia sclerotiorum (A) and (B)Sclerotinia sclerotiorum) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) And/or Gray grape cells (Botrytis cinerea）。
3. The use of streptomyces rochei YC117 as set forth in claim 1 for the preparation of a nematode insecticide.
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| CN116042461A (en) * | 2022-11-29 | 2023-05-02 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Streptomyces rohder and application thereof in disease prevention and control of medlar |
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| CN116042461B (en) * | 2022-11-29 | 2023-09-15 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Streptomyces rohder and application thereof in disease prevention and control of medlar |
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