CN109182213B - Sphingomonas X1-8 in tobacco planting soil and application thereof in prevention and treatment of tobacco black shank - Google Patents
Sphingomonas X1-8 in tobacco planting soil and application thereof in prevention and treatment of tobacco black shank Download PDFInfo
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Abstract
The invention discloses a sphingomonas mobilis X1-8 for tobacco planting and application thereof in prevention and treatment of tobacco black shank, wherein the sphingomonas mobilis X1-8 is stored in China center for type culture Collection with the address: wuhan university, Wuhan, China 430072, preservation date of 2018, 08 and 27 months, and preservation number CCTCC NO of M2018569. The sphingomonas X1-8 provided by the invention can be used for preparing a microbial agent for preventing and treating tobacco black shank, and the prepared microbial agent can effectively prevent and treat tobacco black shank and has the characteristics of low use cost, no residue and safety to people and livestock.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a sphingomonas X1-8 for tobacco planting soil and application thereof in prevention and treatment of tobacco black shank.
Background
Tobacco black shank is a soil-borne disease caused by Phytophthora nicotianae (Phytophthora nicotiana) infecting the root and stem base of tobacco. The pathogenic bacteria can damage all cultivated tobaccos such as flue-cured tobaccos, air-cured tobaccos, sun-cured tobaccos, burley tobaccos, aromatic tobaccos and the like, and has extremely strong destructive power. The average incidence rate of the tobacco black shank is 10 to 15 percentThe disease rate of the field can reach 75 percent, and the serious tobacco field is even in failure. The disease incidence area of tobacco black shank in China every year is about 76372hm2The economic loss is as high as 1.3 billion yuan; tobacco black shank occurs in all large smoke areas all over the world, and particularly rampant in temperate zones, subtropical zones and tropical smoke areas, which causes huge loss to the tobacco planting industry.
At present, the prevention and control of the tobacco black shank are still mainly chemical prevention and control, but biological prevention and control are emphasized due to the unique safety, and the development of new biological control resources and the research and development of efficient biological pesticides are particularly important for the prevention and control of the tobacco black shank and the production of green tobacco leaves. Compared with the traditional chemical pesticide, the microbial pesticide has many advantages, especially obvious advantages in the aspects of environmental compatibility and safety, such as no pollution, no residue, no harm to human and livestock, and the like. Pesticides of microbial origin are considered as effective alternatives to traditional chemical pesticides.
Disclosure of Invention
The invention provides a tobacco-planting soil sphingomonas X1-8 and application thereof in tobacco black shank prevention and treatment, which is used for providing a strain developed by a microbial source microbial inoculum applied to tobacco black shank prevention and treatment, and avoiding or reducing the problems of drug resistance, environmental pollution and human health threat caused by unreasonable use of chemical pesticides.
The invention discloses a tobacco planting soil sphingomonas X1-8, which is preserved in China center for type culture Collection with the address: wuhan university, 430072, preservation date of 2018, 08 and 27 months, and preservation number CCTCC NO: M2018569.
The sphingomonas nicotinovorans X1-8, wherein the culture method of the sphingomonas nicotinovorans X1-8 strain comprises the following steps:
obtaining sphingomonas X1-8 strain from flue-cured tobacco soil;
inoculating the strain to R2Culturing on the slant of the culture medium at 30 ℃ for 7 days to obtain slant seeds;
r for inoculating slant seeds into triangular flask2A in liquid medium at 30 ℃ and 180rCulturing for 5 days under the condition of the rotation frequency of the pm shaking table to obtain liquid seeds;
inoculating liquid seeds into R in a fermentation tank according to the volume proportion of 3%2In the liquid culture medium A, fermenting for 96 hours to obtain the fermentation liquor of the sphingomonas X1-8 strain of the tobacco-planting soil; the culture conditions in a 1000 l fermenter were controlled as follows: the temperature was 30 ℃ and the stirring speed was 150 rpm.
The invention discloses an application of sphingomonas X1-8 in tobacco black shank prevention, preferably, when tobacco seedlings grow to 5-6 leaves, the roots of the tobacco seedlings are stained with sphingomonas X1-8 powder in the tobacco planting soil, and the using amount is 5 g/plant;
the preparation method of the sphingomonas X1-8 powder for the tobacco planting soil comprises the following steps:
centrifuging the fermentation liquor of the sphingomonas X1-8 strain of the tobacco-planting soil at 10000rpm for 10min, and removing the supernatant to obtain the sphingomonas X1-8 strain of the tobacco-planting soil;
uniformly mixing the sphingomonas nicotinovorans X1-8 thallus with diatomite according to the mass ratio of 1:4, then drying or airing at the temperature of below 65 ℃ until the water content is less than 5%, grinding to obtain sphingomonas nicotinovorans X1-8 powder, wherein the viable count content of the sphingomonas nicotinovorans X1-8 strain in the sphingomonas nicotinovorans X1-8 powder is 8.5 multiplied by 109More than one per gram.
The sphingomonas X1-8 for tobacco planting soil and the application thereof in the prevention and treatment of the black shank of tobacco have the following advantages:
1. the prevention effect of the microbial inoculum prepared by the sphingomonas X1-8 strain on the tobacco black shank can reach more than 65 percent.
2. The microbial agent prepared by using the sphingomonas X1-8 strain in the tobacco-planting soil can efficiently prevent and treat the tobacco black shank, and has the characteristics of low use cost, no residue and safety to people and livestock.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail, so that how to implement the technical means for solving the technical problems and achieving the technical effects of the present invention can be fully understood and implemented.
The inventor obtains Sphigomonas tabacum X1-8 strain in flue-cured tobacco soil in Kunming city of Yunnan province in 2017, and finds that the Sphigomonas tabacum X1-8 strain has extremely strong bacteriostatic activity on hypha growth of phytophthora nicotianae, is a potential biocontrol strain for researching and developing biological pesticides for tobacco black shank, and the antifungal activity of the bacterium and the application of the bacterium in the control of soil-borne diseases of tobacco are not reported.
The invention discloses a tobacco planting soil sphingomonas X1-8, which is preserved in China center for type culture Collection with the address: wuhan university, 430072, preservation date of 2018, 08 and 27 months, and preservation number CCTCC NO: M2018569.
The sphingomonas nicotinovorans X1-8, wherein the culture method of the sphingomonas nicotinovorans X1-8 strain comprises the following steps:
obtaining sphingomonas X1-8 strain from flue-cured tobacco soil;
inoculating the strain to R2Culturing on the slant of the culture medium at 30 ℃ for 7 days to obtain slant seeds;
r for inoculating slant seeds into triangular flask2Culturing in liquid culture medium at 30 deg.C and shaking table rotation frequency of 180rpm for 5 days to obtain liquid seed;
inoculating liquid seeds into R in a fermentation tank according to the volume proportion of 3%2In the liquid culture medium A, fermenting for 96 hours to obtain the fermentation liquor of the sphingomonas X1-8 strain of the tobacco-planting soil; the culture conditions in a 1000 l fermenter were controlled as follows: the temperature was 30 ℃ and the stirring speed was 150 rpm.
The invention discloses an application of sphingomonas X1-8 in tobacco black shank prevention, preferably, when tobacco seedlings grow to 5-6 leaves, the roots of the tobacco seedlings are stained with sphingomonas X1-8 powder in the tobacco planting soil, and the using amount is 5 g/plant;
the preparation method of the sphingomonas X1-8 powder for the tobacco planting soil comprises the following steps:
centrifuging the fermentation liquor of the sphingomonas X1-8 strain of the tobacco-planting soil at 10000rpm for 10min, and removing the supernatant to obtain the sphingomonas X1-8 strain of the tobacco-planting soil;
uniformly mixing the sphingomonas nicotinovorans X1-8 thallus with diatomite according to the mass ratio of 1:4, then drying or airing at the temperature of below 65 ℃ until the water content is less than 5%, grinding to obtain sphingomonas nicotinovorans X1-8 powder, wherein the viable count content of the sphingomonas nicotinovorans X1-8 strain in the sphingomonas nicotinovorans X1-8 powder is 8.5 multiplied by 109More than one per gram.
The sphingomonas X1-8 strain is a gram-negative aerobic bacterium in the plant-tobacco soil, and the strain is shown in the formula R2A medium (0.5 g of yeast powder, 0.5g of tryptone, 0.5g of casamino acid, 0.5g of glucose, 0.5g of soluble starch, 0.3g of dipotassium phosphate, 0.05g of magnesium sulfate heptahydrate, 0.3g of sodium pyruvate, 15g of agar and water to 1,000ml and pH 7.0) is cultured for 7 days at 30 ℃, the diameter of a bacterial colony is 2-3mm, and the bacterial colony is yellow, smooth, round and convex in the middle; the cells are round rods with width of 0.4-0.5 μm and length of 0.7-1.6 μm, and have flagella and motility. The strain grows at the temperature of 25-40 ℃, and the optimal growth temperature is 30-35 ℃; can grow in the pH range of 6.0-8.0, and the optimum growth pH is 7.0. The sequence obtained by PCR amplification of 16S rRNA gene of X1-8 strain by using universal primers 27f and 1492r in GeneBank public database is MF370621, and the sequence is the main molecular characteristic basis for identifying the strain. The main fatty acid of the X1-8 strain is 7-octadecenoic acid and 6-octadecenoic acid, the total content of the two accounts for 60.1%, and the main chemical characteristic basis for identifying the strain is provided.
The present invention will be described in further detail with reference to examples. The microbial agent in the embodiment can be prepared by a conventional microbial fermentation method and a conventional microbial agent preparation method.
1. Culture of sphingomonas X1-8 strain in tobacco-planting soil and preparation of microbial inoculum
X1-8 strain test tube slant seed culture: inoculating the strain to R2Culturing on the slant of the culture medium at 30 ℃ for 7 days to obtain slant seeds;
liquid seed culture of the X1-8 strain:r for inoculating slant seeds into triangular flask2In the liquid culture medium A, carrying out shake culture for 5 days at 30 ℃ and 180rpm to obtain liquid seeds;
the X1-8 strain fermentor was used for mass culture: inoculating liquid seed into R in fermentation tank at 3% (V/V)2A liquid culture medium, the culture conditions in a 1000L fermenter were controlled as follows: the temperature is 30 ℃, the stirring speed is 150rpm, and the fermentation time is 96 hours;
preparation of X1-8 powder: centrifuging at 10000rpm for 10min to obtain X1-8 strain fermentation liquid, removing supernatant to obtain X1-8 thallus, mixing thallus with diatomaceous earth at a mass ratio of 1:4, oven drying or air drying at 65 deg.C until the water content is less than 5%, and grinding to obtain powder with viable count of X1-8 strain of 8.5 × 109More than one per gram.
2. Greenhouse test for control effect of sphingomonas X1-8 microbial inoculum on tobacco black shank in tobacco planting soil
Cultivation of phytophthora nicotianae (p. nicotianae): infected tobacco plants are collected from tobacco fields in Puer city in Yunnan province, diseased tissues are taken and inoculated on a PDA culture medium plate after surface disinfection according to a conventional method, fresh mycelia are picked up and inoculated in a PDB (PDA without agar) culture medium after culture is carried out for 5 days at 28 ℃, and shaking culture is carried out for 5 days at 150rpm at 28 ℃ for later use.
Reagent to be tested: x1-8 powder with viable count of 8.5 × 109One/g, prepared according to the above-described method of the present invention; the contrast chemical pesticide is 58% metalaxyl manganese zinc wettable powder produced by Xian Dingsheng biological chemical company Limited.
The test method comprises the following steps: culturing flue-cured tobacco seeds 'Yunyan 87' by adopting a conventional floating seedling culture technology, dipping roots of the tobacco seeds with 5 g/plant of X1-8 powder when the tobacco seedlings grow to 5-6 leaves, then transplanting the tobacco seeds into a flowerpot with the diameter of 15cm and filled with 500g of soil (sand: humus: 1), and inoculating 5ml of phytophthora nicotianae culture solution to tobacco roots. For a positive control, the X1-8 powder is replaced by a mixture of 1g of 58% metalaxyl manganese zinc wettable powder and 4g of diatomite; blank control X1-8 powder was replaced with 5g of diatomaceous earth. Three replicates per treatment, 5 replicates per replicate; the management of fertilizer and water is carried out according to a conventional method.
And (4) calculating the control effect: the tobacco seedlings treated by the method are randomly arranged and placed in a greenhouse, the incidence rate of the tobacco black shank treated by the method is counted after the tobacco seedlings grow for 90 days, and disease classification, disease index and prevention effect are calculated according to the method introduced in GB/T23222-2008 tobacco pest classification and investigation method.
And (3) test results: as can be seen from the table 1, the control effect of the X1-8 powder on the tobacco black shank is 79.8 percent and the control effect of 58 percent of metalaxyl manganese zinc is 80.2 percent under the greenhouse condition; the difference between the two is not significant.
TABLE 1 prevention of tobacco black shank by X1-8 powder and 58% metalaxyl manganese zinc under greenhouse and field conditions
Note: the letters after the numerical values in the same column are the same, which indicates that the difference is not obvious; otherwise, the difference is significant.
3. Field test of effect of sphingomonas X1-8 microbial inoculum on prevention of tobacco black shank in tobacco planting soil
Reagent to be tested: x1-8 powder with viable count of 8.5 × 109One/g, prepared according to the above-described method of the present invention; the contrast chemical pesticide is 58% metalaxyl manganese zinc wettable powder produced by Xian Dingsheng biological chemical company Limited.
Flue-cured tobacco variety: and (4) cloud smoke 87.
Test site: puer Cijing Gu county Yongping Town tobacco field with serious black shank disease.
The test method comprises the following steps: the tobacco field is divided into 9 cells, and the area of each cell is 50 square meters. The experiment was run for three treatments: treating with X1-8 powder, treating with 58% metalaxyl manganese zinc, and treating with clear water. Each treatment was repeated three times, each treatment was first randomized. The tobacco is grown and transplanted according to the conventional method, and 90 tobacco plants are planted in each cell. The application of the drug is started 15 days after the transplantation. For each flue-cured tobacco treated by X1-8 powder, 5g X1-8 powder is diluted in 500ml tap water and one tobacco root is irrigated. For each flue-cured tobacco treated with 58% metalaxyl manganese zinc, a mixture of 1g and 4g of diatomaceous earth was used in place of the X1-8 powder; for each flue-cured tobacco treated with a clear water control, 4g of diatomaceous earth was used in place of the X1-8 powder. The management of fertilizer and water is carried out according to a conventional method.
And (4) calculating the control effect: after the medicine is applied, the tobacco plants are grown for 60 days, the incidence rate of the tobacco black shank of each treatment is counted, and disease classification, disease index and prevention effect are calculated according to the method introduced in GB/T23222-2008 tobacco pest classification and investigation method.
And (3) test results: as can be seen from the table 1, the control effect of the X1-8 powder on the tobacco black shank is 72.4 percent and the control effect of 58 percent of metalaxyl manganese zinc is 73.6 percent under the field condition; the difference between the two is not significant. The practical application shows that: the microbial inoculum prepared by the X1-8 strain can effectively prevent and treat the tobacco black shank.
While the foregoing description shows and describes several preferred embodiments of this invention, it is to be understood, as noted above, that this invention is not limited to the forms disclosed herein, but is not intended to be exhaustive or to exclude other embodiments and may be used in various other combinations, modifications, and variations within the scope of the inventive concept, as may be realized by the teachings set forth above or as may be learned by the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. Sphingosine monad (A) for tobacco planting soilSphigomonastabacisoli) The application of X1-8 in the control of tobacco black shank is characterized in that: deposited in China center for type culture Collection, address: wuhan university, Wuhan, China 430072, preservation date of 2018, 08 and 27 months, and preservation number CCTCC NO of M2018569.
2. The use of claim 1, wherein when the tobacco seedling grows to 5-6 leaves, the root of the tobacco seedling is stained with sphingomonas X1-8 powder of the tobacco planting soil in an amount of 5 g/plant;
the preparation method of the sphingomonas X1-8 powder for the tobacco planting soil comprises the following steps:
centrifuging the fermentation liquor of the sphingomonas X1-8 strain of the tobacco-planting soil at 10000rpm for 10min, and removing the supernatant to obtain the sphingomonas X1-8 strain of the tobacco-planting soil;
uniformly mixing the sphingomonas nicotinovorans X1-8 thallus with diatomite according to the mass ratio of 1:4, then drying or airing at the temperature of below 65 ℃ until the water content is less than 5%, grinding to obtain sphingomonas nicotinovorans X1-8 powder, wherein the viable count content of the sphingomonas nicotinovorans X1-8 strain in the sphingomonas nicotinovorans X1-8 powder is 8.5 multiplied by 109More than one per gram.
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