CN116083328B - Siamese bacillus, microbial agent and application thereof - Google Patents
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Abstract
The application is applicable to the technical field of microorganisms and provides a Siamese bacillus, a microbial agent and application thereof. The application provides Siamese bacillusBacillus siamensis) The ZXKN01 strain can resist pathogenic fungi, pathogenic bacteria and North root-knot nematodes, and the microbial agent prepared by the ZXKN01 strain has a control effect on the North root-knot nematodes on ginseng traditional Chinese medicinal materials of more than 70%, and has a control effect on the composite diseases of the North root-knot nematodes of more than 60%. The microbial agent prepared by the ZXKN01 strain can effectively prevent and treat the northern root knot nematode disease on ginseng traditional Chinese medicinal materials, has a certain prevention effect on soil-borne fungus diseases and bacterial diseases, and has the characteristics of low use cost, no residue and safety to people and livestock.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to a bacillus siamensis, a microbial agent and application thereof.
Background
The root-knot nematode compound disease refers to a disease caused by that root-knot nematodes living in the same ecological niche and soil-borne pathogenic bacteria synergistically infect the same crop. The wound caused by the root system infection of the root-knot nematode second-instar larvae is beneficial to the infection of various soil-borne pathogenic fungi and pathogenic bacteria; therefore, the root-knot nematode compound diseases commonly occur and are seriously damaged on various crops. The root knot nematode compound disease is reported for the first time in the composite infection of cotton by the root knot nematode and fusarium oxysporum. Heretofore, compound diseases caused by synergistic infection of root-knot nematodes with various plant pathogenic fungi and pathogenic bacteria are widely reported in crops such as tomatoes, traditional Chinese medicinal materials, tobacco, alfalfa, cotton, bananas, coffee, beans and the like, and have serious harm and huge loss.
Root knot nematode disease on ginseng traditional Chinese medicinal materials is caused by northern root knot nematode infection, and root knot nematode compound diseases are extremely serious. For example, compound diseases of the root-knot nematode of pseudo-ginseng caused by compound infection of the root-knot nematode in the north with the pathogenic bacteria pseudomonas, aureobacterium indoxylum producing and pathogenic fungi phytophthora, phoma herbicola are very common. The compound disease of American ginseng and ginseng root-knot nematode caused by the compound infection of the North square root-knot nematode and pathogenic fungi such as fusarium, aschersonia, phoma herbicola and the like has large damage area and serious economic loss.
At present, related researches on prevention and treatment of northern root-knot nematodes on ginseng traditional Chinese medicinal materials are few, and the prevention and treatment effect is required to be improved.
Disclosure of Invention
The embodiment of the application aims to provide the Siamese bacillus, and aims to provide the Siamese bacillus capable of effectively preventing and controlling the North root-knot nematodes and the compound diseases thereof on ginseng traditional Chinese medicinal materials.
The embodiment of the application is realized in such a way that the bacillus siamensis is bacillus siamensis @Bacillus siamensis) ZXKN01 is preserved in China general microbiological culture Collection center (CGMCC) No.25830, and the preservation date is 2022, 09 and 28.
Another object of the embodiments of the present application is a microbial inoculant comprising the Bacillus siamensis strain described aboveBacillus siamensis）ZXKN01。
Another object of the embodiments of the present application is to provide a method for preparing the microbial agent, including:
the Siamese bacillus is treatedBacillus siamensis) Inoculating ZXKN01 strain on the slant of the culture medium for culturing to obtain slant seeds;
inoculating the slant seeds into a liquid culture medium for culture to obtain liquid seeds;
inoculating the liquid seeds into a culture medium in a fermentation tank according to the volume ratio of 3-5% for fermentation culture to obtain thalli and metabolites thereof;
mixing the thalli and the metabolites thereof with soluble corn starch, and performing spray drying treatment to obtain the microbial agent.
Another object of the embodiment of the present application is to use the bacillus siamensis or the microbial agent in preventing and treating northern root knot nematode disease on ginseng traditional Chinese medicinal materials.
Another object of the embodiment of the present application is to use the bacillus siamensis or the microbial agent in controlling compound diseases of root-knot nematodes of ginseng traditional Chinese medicinal materials.
The embodiment of the application provides Siamese bacillusBacillus siamensis) The ZXKN01 strain can resist pathogenic fungi, pathogenic bacteria and North root-knot nematodes,the microbial agent prepared by the ZXKN01 strain has the effect of preventing the northern root-knot nematode disease on ginseng traditional Chinese medicinal materials of more than 70 percent and has the effect of preventing the northern root-knot nematode compound disease of more than 60 percent. The microbial agent prepared by the ZXKN01 strain can effectively prevent and treat the northern root knot nematode disease on ginseng traditional Chinese medicinal materials, has a certain prevention effect on soil-borne fungus diseases and bacterial diseases, and has the characteristics of low use cost, no residue and safety to people and livestock.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The application takes the composite disease pathogen of the North root knot nematode on the ginseng traditional Chinese medicine as a target, and screens Siamese bacillusBacillus siamensis) ZXKN01 strain can resist pathogenic fungi, pathogenic bacteria and North root-knot nematode. At present, not only the strain of the Siamese bacillus with three pathogens resistance and the application thereof are not reported, but also the application of the Siamese bacillus in preventing and controlling the North root-knot nematode on the ginseng traditional Chinese medicinal materials is not reported.
The application provides a Siamese bacillusBacillus siamensis) ZXKN01 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.25830 and a preservation date of 2022, 09 and 28 days, and the preservation unit address is China academy of China microbiological study, national institute No. 3, north Chen West Lu 1, korea, beijing.
In the research process of screening nematode biocontrol bacteria, 45 Siamese bacillus strains are separated from two-instar larvae (J2), eggs and epiphytes of female worms of root-knot nematodes from different sources. Taking northern root-knot nematode J2 infecting American ginseng as a target to obtain a strain 12 with the mortality rate to J2 being more than 70%; then taking phytophthora capsici and pseudomonas fluorescens serving as pathogenic bacteria as targets, determining the antagonism of the 12 strain of bacillus siamensis LB fermentation liquor to the two pathogens, and finding that the 12 strain of bacillus siamensis LB fermentation liquor is only separated from American ginsengSiamese bacillus on the North square root knot nematode J2Bacillus siamensis) ZXKN01 strain showed good biocontrol activity against three pathogens (Table 1).
Wherein, siamese bacillus isBacillus siamensis) ZXKN01 Strain at R 2 Culturing on the culture medium A at 37deg.C for 3 days to obtain colony with diameter of 2-4 mm, and has the advantages of liquid viscosity, translucency and regular edge; the strain can grow at 5-55deg.C and pH 4.5-9.0, and the strain grows best at 37 deg.C and pH 6-7. Siamese bacillusBacillus siamensis) The ZXKN01 strain is gram-positive bacteria, aerobic or facultative anaerobic bacteria, thallus rod-shaped, and has a cell width of 0.25-0.65 mm and a length of 1.2-3.6 mm. The physiological and biochemical characteristics of ZXKN01 strain are that whole cell fatty acid analysis shows that the main fatty acid is antais-C 15:0 And antais-C 17:0 . Cell-whole polar lipid analysis revealed that it contained biphospholipid glycerol, phosphatidylglycerol, phosphatidylethanolamine, lysyl phosphatidylglycerol, glycolipids and other unknown lipids; the physicochemical characteristics are the main chemical characteristic basis for identifying ZXKN 01. The sequence obtained by amplifying the 16S rRNA gene of ZXKN01 strain by PCR using the universal primers 27f and 1492r is the sequence number OP459296 in the GeneBank public database, and the sequence is the main molecular characteristic basis for identifying the strain.
In addition, the microbial agent provided by the application can be prepared according to a conventional microbial fermentation method and a conventional microbial agent preparation method.
Examples of certain embodiments of the present application directed to the acquisition of the Siamese bacillus, microbial agents and their use are given below, and are not intended to limit the scope of the present invention.
EXAMPLE 1 Siamese bacillusBacillus siamensis) Obtaining ZXKN01 Strain
J2, eggs and females of the root-knot nematodes are separated from pseudo-ginseng, american ginseng and ginseng root nodules infected by the root-knot nematodes, the J2, eggs and females are respectively inoculated on LB culture medium plates, and 145 single colonies of nematode-attached bacteria are obtained according to a traditional streak separation method after the culture is carried out for 3 days at 37 ℃. Culturing the 145 strains of bacteria with LB liquid medium at 37 ℃ and 150 rpm for 3 days to obtain bacterial fermentation liquor, and measuring the mortality of each bacterial fermentation liquor to American ginseng northern root-knot nematode J2, wherein the mortality of 12 strains to J2 is more than 70 percent (table 1); and then taking phytophthora capsici which is a pathogenic fungus of the American ginseng northern root-knot nematode compound disease and pseudomonas fluorescens which is a pathogenic bacterium as targets, and determining the antagonism of the 12 siamese bacillus LB fermentation liquor to the two pathogens. The results show that 5 strains (ZXKN 01, ZXKN04, ZXKN07, ZXKN09 and ZXKN 11) of the 12 strains can simultaneously resist three target pathogens, wherein the activity of the ZXKN01 strain is best, the mortality rate of the strain to the northern root-knot nematode J2 is 86.3%, the growth inhibition rate to phytophthora capsici is 57.2%, and the inhibition zone to the pseudomonas fluorescens is 5.7 mm. There were 4 strains (ZXKN 02, ZXKN05, ZXKN08, ZXKN 10) that were both resistant to both pathogens, the remaining 3 strains (ZXKN 03, ZXKN06, ZXKN 12) were only antagonistic to J2 and were not active against both target pathogenic fungi and bacteria (table 1).
TABLE 1 determination of antagonism of Coptoterus root knot nematode epiphyte bacteria against North Coptoterus root knot nematode Compound disease pathogen
EXAMPLE 2 cultivation of ZXKN01 Strain and preparation of microbial agent
ZXKN01 strain test tube slant seed culture: the strain is inoculated on the inclined plane of a conventional LB culture medium, and the inclined plane seeds are obtained after 3 days of culture at 37 ℃.
Liquid seed culture of ZXKN01 strain: the slant seeds were inoculated into a triangular flask containing LB liquid medium, and shake-cultured at 37℃and 120 rpm for 3 days to obtain liquid seeds.
Large-scale culture of ZXKN01 strain fermentation tanks: the liquid seeds are inoculated into LB culture medium in a fermentation tank according to the proportion of 3-5% (V/V), and the culture condition in the fermentation tank of 500-1000 liters is controlled as follows: the temperature is 32-37 ℃, the stirring speed is 150-180 rpm, and the fermentation time is 48-72 hours.
Preparation of a microbial inoculum of ZXKN01 strain: the bacterial strain ZXKN01 and its metabolite are mixed with proper amount of soluble corn starch, and the bacterial agent is obtained through spray drying, the water content of the bacterial agent is controlled below 5%, and the effective viable count of the bacterial strain ZXKN01 in the bacterial agent is above 10 hundred million CFU/g.
Application example 1 ZXKN01 microbial agent for controlling root-knot nematode and composite disease thereof
Test site: american ginseng planting base in Wendelia of WiHai in Shandong province is a land block where northern root-knot nematodes and compound diseases thereof occur seriously.
Test crop: american ginseng.
Test agent: ZXKN01 microbial agent, the viable count content is 12 hundred million CFU/gram, prepare according to the above-mentioned method of the invention; the control pesticide for preventing and controlling the root-knot nematode is 10% fosthiazate granules (produced by Shandong province United agricultural chemical industry Co., ltd.), the control pesticide for preventing and controlling the soil fax diseases is 30% hymexazol aqueous solution (Fangfang Hua Nuo biological technology Co., ltd.), and the control pesticide for preventing and controlling the soil bacterial diseases is 20% thiabendazole suspension (Zhanjiang Longwan chemical Co., ltd.). American ginseng is cultivated according to the conventional method, and is used as a medicine during transplanting.
The test method comprises the following steps: the following three treatments were set up, 3 replicates per treatment, and 0.3 mu per replicate.
Treatment 1: ZXKN01 microbial agent, 1500 g/mu, taking 500 g microbial agent when transplanting, diluting with 200 kg water, and dipping roots; 1000 g of microbial inoculum is taken 60 days after transplanting, diluted by 800 kg of water and root irrigation is carried out.
Treatment 2: 10% fosthiazate granules (1500 g/mu) are spread on a carriage surface and uniformly mixed into a cultivation layer during soil preparation, and 30% hymexazol aqueous solution (500 ml/mu) and 20% thiabendazole copper suspension (500 ml/mu) are respectively diluted according to instructions and then root irrigation is carried out after 7 days of transplanting.
Treatment 3: blank control without any pesticide applied.
Investigation and statistical methods: at 12 months after transplanting, 100 American ginseng plants are randomly taken for each treatment, and plant disease classification, disease index calculation and prevention effect are respectively carried out on the root-knot nematode disease and the compound disease according to a method described in literature (Huang et al 2015,Pest Management Science, 71:415-422).
Test results: table 2 shows that the treatment 1 applied 1.5 kg of ZXKN01 bacterial agent with the concentration of 12 hundred million CFU/g has the control effects of 79.36% and 62.05% on American ginseng northern root-knot nematode disease and northern root-knot nematode compound disease respectively. In the treatment 2, chemical pesticides respectively aiming at root-knot nematodes, pathogenic fungi and pathogenic bacteria are applied simultaneously, and the control effects on American ginseng northern root-knot nematode diseases and northern root-knot nematode compound diseases are 81.44% and 69.87%, respectively. The two treatments have no obvious difference on the prevention effect of the northern root knot nematode disease, and the prevention effect of the ZXKN01 microbial agent on the compound disease is obviously lower than that of chemical pesticides.
TABLE 2 control of ZXKN01 microbial inoculant on American ginseng northern root-knot nematode and composite diseases thereof
Note that: the letters after the same column of numbers are the same, which indicates no significant difference, otherwise the difference is significant.
Application example 2 ZXKN01 microbial agent for controlling root-knot nematode and compound disease of pseudo-ginseng
Test site: the planting area of pseudo-ginseng in Wenshan, yunnan province is a land block where northern root knot nematode and compound diseases thereof are seriously generated.
Test crop: pseudo-ginseng.
Test agent: ZXKN01 microbial agent, the viable count content is 11 hundred million CFU/gram, prepare according to the above-mentioned method of the invention; the control pesticide for preventing and controlling the root-knot nematode is 10% fosthiazate granules (produced by Shandong province United agricultural chemical industry Co., ltd.), the control pesticide for preventing and controlling the soil fax diseases is 30% hymexazol aqueous solution (Fangfang Hua Nuo biological technology Co., ltd.), and the control pesticide for preventing and controlling the soil bacterial diseases is 20% thiabendazole suspension (Zhanjiang Longwan chemical Co., ltd.). Raising seedlings of pseudo-ginseng according to a conventional method, and taking medicines during transplanting.
The test method comprises the following steps: the following three treatments were set up, 3 replicates per treatment, and 0.3 mu per replicate.
Treatment 1: ZXKN01 microbial agent, 1500 g/mu, taking 500 g microbial agent when transplanting, diluting with 200 kg water, and dipping roots; 1000 g of microbial inoculum is taken 60 days after transplanting, diluted by 800 kg of water and root irrigation is carried out.
Treatment 2: 10% fosthiazate granules (1500 g/mu) are spread on a carriage surface and uniformly mixed into a cultivation layer during soil preparation, and 30% hymexazol aqueous solution (500 ml/mu) and 20% thiabendazole copper suspension (500 ml/mu) are respectively diluted according to instructions and then root irrigation is carried out after 7 days of transplanting.
Treatment 3: blank control without any pesticide applied.
Investigation and statistical methods: at 12 months after transplanting, 100 pseudo-ginseng plants are randomly taken for each treatment, and plant disease classification, disease index calculation and prevention effect are respectively carried out on the root-knot nematode disease and the compound disease according to a method described in literature (Huang et al 2015,Pest Management Science, 71:415-422).
Test results: table 3 shows that the microbial inoculum of ZXKN01 with the concentration of 11 hundred million CFU/g is applied to each mu in the treatment 1, and the control effect on the compound diseases of the pseudo-ginseng North root-knot nematode and the North root-knot nematode is 77.15 percent and 62.45 percent respectively. In the treatment 2, chemical pesticides respectively aiming at root-knot nematodes, pathogenic fungi and pathogenic bacteria are applied simultaneously, and the control effects on the pseudo-ginseng North root-knot nematode disease and the North root-knot nematode compound disease are respectively 78.34% and 63.35%. The two treatments have no obvious difference in the prevention effect on the northern root knot nematode and the compound diseases.
TABLE 3 control of ZXKN01 microbial inoculant on pseudo-ginseng root-knot nematode and composite diseases thereof
Note that: the letters after the same column of numbers are the same, which indicates no significant difference, otherwise the difference is significant.
Application example 3 ZXKN01 microbial agent control test on root-knot nematode and composite diseases thereof
Test site: the ginseng planting area in Fusong county in Jilin province, white mountain city, and the test area is a plot of severe occurrence of northern root knot nematode disease and compound diseases thereof.
Test crop: ginseng.
Test agent: ZXKN01 microbial agent, the viable count content is 10 hundred million CFU/gram, prepare according to the above-mentioned method of the invention; the control pesticide for preventing and controlling the root-knot nematode is 10% fosthiazate granules (produced by Shandong province United agricultural chemical industry Co., ltd.), the control pesticide for preventing and controlling the soil fax diseases is 30% hymexazol aqueous solution (Fangfang Hua Nuo biological technology Co., ltd.), and the control pesticide for preventing and controlling the soil bacterial diseases is 20% thiabendazole suspension (Zhanjiang Longwan chemical Co., ltd.). The ginseng is cultivated according to the conventional method, and is used as a medicine during transplanting.
The test method comprises the following steps: the following three treatments were set up, 3 replicates per treatment, and 0.3 mu per replicate.
Treatment 1: ZXKN01 microbial agent, 1500 g/mu, taking 500 g microbial agent when transplanting, diluting with 200 kg water, and dipping roots; 1000 g of microbial inoculum is taken 60 days after transplanting, diluted by 800 kg of water and root irrigation is carried out.
Treatment 2: 10% fosthiazate granules (1500 g/mu) are spread on a carriage surface and uniformly mixed into a cultivation layer during soil preparation, and 30% hymexazol aqueous solution (500 ml/mu) and 20% thiabendazole copper suspension (500 ml/mu) are respectively diluted according to instructions and then root irrigation is carried out after 7 days of transplanting.
Treatment 3: blank control without any pesticide applied.
Investigation and statistical methods: at 12 months after transplanting, 100 ginseng plants are randomly taken for each treatment, and plant disease classification, disease index calculation and prevention effect are respectively carried out on the root-knot nematode disease and the compound disease according to a method described in literature (Huang et al 2015,Pest Management Science, 71:415-422).
Test results: table 4 shows that the microbial inoculum of ZXKN01 with the concentration of 10 hundred million CFU/g is applied to each mu in the treatment 1, and the control effect on the compound diseases of the ginseng North square root knot nematode and the northern root knot nematode is 76.18 percent and 60.38 percent respectively. In the treatment 2, chemical pesticides respectively aiming at root-knot nematodes, pathogenic fungi and pathogenic bacteria are applied simultaneously, and the control effects on the compound diseases of the root-knot nematodes and the northern root-knot nematodes of the ginseng are 74.97% and 66.99%, respectively. The two treatments have no obvious difference on the prevention effect of the northern root knot nematode disease, and the prevention effect of the ZXKN01 microbial agent on the compound disease is obviously lower than that of chemical pesticides.
TABLE 4 control of ZXKN01 microbial inoculant on root-knot nematode and composite diseases thereof
Note that: the letters after the same column of numbers are the same, which indicates no significant difference, otherwise the difference is significant.
In summary, the embodiment of the application utilizes the Siamese bacillusBacillus siamensis) The microbial agent prepared by the ZXKN01 strain has the effects of preventing and controlling the northern root knot nematode on the ginseng traditional Chinese medicinal materials above 70 percent and preventing and controlling the northern root knot nematode compound diseases above 60 percent, and can be used for effectively preventing and controlling the northern root knot nematode on the ginseng traditional Chinese medicinal materials, has certain effects of preventing and controlling soil-borne fungus diseases and bacterial diseases, and has the characteristics of low use cost, no residue and safety to people and livestock.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. A Siamese bacillus is characterized in that the Siamese bacillus is a Siamese bacillus @Bacillus siamensis) ZXKN01 is preserved in China general microbiological culture Collection center (CGMCC) No.25830, and the preservation date is 2022, 09 and 28.
2. A microbial agent is characterized in that,the microbial agent comprises the Siamese bacillus as claimed in claim 1Bacillus siamensis）ZXKN01。
3. A method of preparing the microbial agent of claim 2, comprising:
the Siamese bacillus is treatedBacillus siamensis) Inoculating ZXKN01 strain on the slant of the culture medium for culturing to obtain slant seeds;
inoculating the slant seeds into a liquid culture medium for culture to obtain liquid seeds;
inoculating the liquid seeds into a culture medium in a fermentation tank according to the volume ratio of 3-5% for fermentation culture to obtain thalli and metabolites thereof;
mixing the thalli and the metabolites thereof with soluble corn starch, and performing spray drying treatment to obtain the microbial agent.
4. The method for producing a microbial agent according to claim 3, wherein the conditions for fermentation culture are a temperature of 32 to 37℃and a stirring speed of 150 to 180 rpm, and the fermentation time is 48 to 72 hours.
5. The method for producing a microbial agent according to claim 3, wherein the water content of the microbial agent is controlled to 5% or less.
6. The method for preparing microbial agent according to claim 3, wherein the microbial agent is bacillus siamensis @Bacillus siamensis) The effective viable count of ZXKN01 strain is above 10 hundred million CFU/gram.
7. Use of a siamese bacillus according to claim 1 or a microbial agent according to claim 2 for preventing the disease of the root-knot nematode of the ginseng plant chinese-medicinal material.
8. Use of the siamese bacillus of claim 1 or the microbial agent of claim 2 for preventing compound diseases of the ginseng Chinese herbal medicine meloidogyne, wherein pathogenic bacteria of the compound diseases of the meloidogyne are phytophthora and pseudomonas fluorescens.
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| CN109182176A (en) * | 2018-09-10 | 2019-01-11 | 青岛秾贝尔生物工程有限公司 | One plant of Siam bacillus and its application in prevention and treatment tomato root-knot eelworm disease |
| CN111778190A (en) * | 2020-07-16 | 2020-10-16 | 山东滇鲁生物科技有限公司 | Brevibacillus brevis for preventing and treating root knot nematode disease and application thereof |
| CN114891670A (en) * | 2022-04-28 | 2022-08-12 | 岭南师范学院 | Bacillus siamensis ZJh with broad-spectrum antibacterial activity and application thereof |
| CN115747100A (en) * | 2022-10-27 | 2023-03-07 | 吉林农业大学 | Siamese bacillus N2 and application thereof in preventing and treating ginseng root rot |
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