CN116286455A - Agrobacterium strain 18A6 and application, product and method thereof - Google Patents
Agrobacterium strain 18A6 and application, product and method thereof Download PDFInfo
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- CN116286455A CN116286455A CN202211444108.0A CN202211444108A CN116286455A CN 116286455 A CN116286455 A CN 116286455A CN 202211444108 A CN202211444108 A CN 202211444108A CN 116286455 A CN116286455 A CN 116286455A
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- agrobacterium
- ralstonia
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- deltaense
- bacterial wilt
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Abstract
The invention relates to an agrobacterium strain 18A6 and application, a product and a method thereof, belonging to the technical field of microorganisms. The invention provides an agrobacterium (Agrobacterium deltaense) strain 18A6, the preservation number of which is CCTCC No: m2020041. The invention also provides application of the agrobacterium tumefaciens (Agrobacterium deltaense) strain 18A6 in preventing and treating bacterial wilt. The invention also provides a bacterial agent based on the agrobacterium (Agrobacterium deltaense) strain 18A6 and a method for preventing and treating bacterial wilt. Experiments prove that the agrobacterium (Agrobacterium tumefaciens) strain 18A6 can effectively maintain the disease index of bacterial wilt-causing tobacco strains at a low level.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to an agrobacterium strain 18A6 and application, a product and a method thereof.
Background
Bacterial wilt is caused by infection with Ralstonia bacteria (Ralstonia), and is an important soil-borne disease of crops. The host range of the Ralstonia is wide, more than 400 crops can be infected, and the host range is heavy in banana, tomato, potato and tobacco. Tobacco bacterial wilt is mainly caused by infection of pseudomonas solanaceae rotunda (r. Pseudosolanoaceum), and the disease commonly occurs in Yangtze river basin and tobacco regions in south of China. The disease occurs in 43 areas (counties) of 12 states of the Yunnan province. Although the tobacco bacterial wilt is serious, the disease is an important factor for restricting the improvement of the yield and quality of tobacco due to the lack of disease-resistant resources and the lack of chemical prevention and control special-effect drugs.
Biological control is receiving extensive attention and research because it is environmentally friendly. 9 biocontrol bacteria, mainly including bactericides such as pseudomonas fluorescens, bacillus amyloliquefaciens, paenibacillus polymyxa, bacillus subtilis and the like, are developed and registered aiming at tobacco bacterial wilt in China, and most bactericides have the capacity of antagonizing Ralstonia. The related patents of the tobacco bacterial wilt biocontrol bacteria reported in China mainly comprise: ralstonia sp.) 56D2 (CN 113502250A), pseudomonas paraflavum (P.paraflavus) DW15 (CN 114934001A), pseudomonas korea (P.koreensis) CLP-23 (CN 111705016A), bacillus amyloliquefaciens (B.amyloliquefaciens) TBA03 (CN 111117936A), paenibacillus polymyxa NX1-4-4 (CN 107365729A), bacillus subtilis biocontrol strain Trb3 (CN 102747013A); the composite microbial inoculum mainly comprises 3 strains of pseudomonas (P.lurida) FGD5-2, (P.koreensis) HCH2-3 and (P.rhodesiae) MTD4-1 combination (CN 112920965A), streptomyces microflavus (S.microflavus) CGMCC 12841, micro Bai Huanglian mold (S.albidoflavus) CGMCC 12842, streptomyces (S.pratenosis) CGMCC 12843 and 1 strain of pseudomonas aeruginosa combination (CN 106754563A), streptomyces griseus, streptomyces lividans, bacillus sand, brevibacterium midwiferum, trichoderma reesei combination (CN 106465734A), trichoderma reesei (Trichoderma ressei) ACCC 30150 and Huang Zhese streptomyces ACCC 40021 combination (CN 103315005A) and the like.
Agrobacterium (agrobacteria) has been used less frequently in agricultural production, only the use of Agrobacterium (agrobacteria) bacteria in combination with other genus bacteria for degrading herbicides, fungicides, pesticides or other crop protection chemicals (CN 106170207 a). The application of the agrobacterium-free bacteria in crop disease control is not seen, nor is the application of the agrobacterium-free bacteria in crop bacterial wilt and tobacco bacterial wilt control.
Disclosure of Invention
Based on the blank existing in the prior art in the field, the invention provides an agrobacterium (Agrobacterium deltaense) 18A6 capable of preventing and treating tobacco bacterial wilt, and application, a product and a method thereof.
The technical scheme of the invention is as follows:
an agrobacterium (Agrobacterium deltaense) strain 18A6, characterized by a collection number of CCTCC No: m2020041.
The preservation number is CCTCC No: use of agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 for controlling bacterial wilt.
The bacterial wilt refers to tobacco bacterial wilt, and is a disease caused by infection of pseudosolanaceae ralstonia (Ralstonia solanacearum) or pseudosolanaceae ralstonia (Ralstonia pseudosolanacearum) or syzygote ralstonia (Ralstonia syzygii).
The agrobacterium (Agrobacterium deltaense) strain 18A6 does not control bacterial wilt by antagonizing or inhibiting solanacearum rogowski (Ralstonia solanacearum) or pseudosolanacearum rogowski (Ralstonia pseudosolanacearum) or syzygospermum rogowski (Ralstonia syzygii).
A microbial inoculum, characterized by comprising: the preservation number is CCTCC No: agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041.
The microbial inoculum also comprises: auxiliary materials.
The microbial inoculum is prepared from the following components in parts by weight: fermentation broth, powder and suspension emulsion.
A method for preventing and treating bacterial wilt is characterized in that the preservation number is CCTCC No: the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 prevents and treats bacterial wilt.
The concentration of the adopted bacteria is 10 7 -10 9 The pathogenic plants were root irrigated with a fermentation dilution of CFU/mL of agrobacterium (Agrobacterium deltaense) strain 18A6 or a suspension of bacterial powder at a bacterial concentration of 133.5 hundred million CFU/g.
The diseased plant refers to: tobacco plants that develop bacterial wilt are infected with Ralstonia solanaceae (Ralstonia solanacearum) or Ralstonia pseudosolanaceae (Ralstonia pseudosolanacearum) or Ralstonia syringiensis (Ralstonia syzygii).
The invention provides an agrobacterium (Agrobacterium deltaense), designated 18A6. One strain identified as Agrobacterium (Agrobacterium deltaense) was isolated from tobacco-planting soil and was deposited at China center for type culture collection (CCTCC, address: eight-channel No. 299 university of Wuhan, hubei province, china center for type culture collection, post code: 430072) for 28 days in month 1 in 2020, and the accession number was CCTCC No: m2020041.
The beneficial effects of the invention are as follows:
1. the disease control mechanism is different: the conventional bacterial wilt biocontrol bacteria screening is carried out through indoor flat plate antagonism primary screening and greenhouse biological test complex screening, but the antibacterial capability is not taken as an evaluation index when the control effect of the agrobacterium (Agrobacterium deltaense) 18A6 is evaluated, the disease control capability is taken as an index, the antagonism of the biocontrol bacteria 18A6 to the Ralstonia is found through the flat plate antagonism capability measurement, the disease control mechanism of the biocontrol bacteria 18A6 is different from that of the traditional antagonism bacteria, and the disease can be prevented through the mechanisms of ecological niche competition, plant disease resistance induction, rhizosphere microbial community regulation and the like;
2. ecological safety: the biocontrol bacteria 18A6 have no inhibition capability on the growth of the Ralstonia, which indicates that the biocontrol bacteria 18A6 do not play a role in controlling diseases mainly based on antibiotic substances. Therefore, when the biocontrol bacteria 18A6 are applied in the field to prevent and treat tobacco bacterial wilt, the resistance of the ralstonia to the biocontrol bacteria 18A6 or the bacterial strain metabolite can not be generated, and the application of the biocontrol bacteria 18A6 has better safety;
3. enriching biocontrol resources: the biocontrol bacteria of the tobacco bacterial wilt are mainly bacillus, pseudomonas, streptomycete and avirulent ralstonia, and agrobacterium (Agrobacterium deltaense) is not reported in biological control of crop diseases, and the agrobacterium (Agrobacterium deltaense) 18A6 provides new biocontrol resources for crop disease control.
The deposit information of the agrobacterium (Agrobacterium deltaense) 18A6 of the invention is as follows:
preservation number: cctccc NO: m2020041;
classification naming: agrobacterium deltaense 18A 18 6;
preservation date: 28 days of 1 month in 2020;
preservation unit: china center for type culture Collection;
preservation address: chinese, wuhan, university of Wuhan.
Drawings
FIG. 1 is a colony morphology and cell morphology of Agrobacterium 18A6 of Experimental example 1 of the present invention cultured for 48 hours.
FIG. 2 is a phylogenetic tree based on whole genome construction of experimental example 3 of the present invention.
FIG. 3 shows the effect of the powder 18A6 of Experimental example 5 on controlling tobacco bacterial wilt
FIG. 4 is a graph showing comparison of morphology of a colony of Agrobacterium 18A6 of Experimental example 6 of the present invention cultured against Ralstonia.
Detailed Description
The technical scheme of the present invention will be described in further detail with reference to specific examples, experimental examples and drawings, but the present invention is not limited to the following technical scheme.
Sources of biological materials
1. The tobacco materials used in experimental examples 2, 4 and 5 are known and commonly used tobacco varieties of safflower Dajinyuan, and are stored in the applicant laboratory and are also commercially available.
2. The ralstonia bacteria used in experimental examples 4, 5 or 6:
the genome sequencing of the Rulstonia pseudosolanaceae (R.pseudonosololanacrum) RS has been completed, the sequence was submitted to the GenBank database, bioproject Number PRJNA594457, no. GenBank assembly accession GCA_018243235.1, and the strain was deposited at the Cantonese province microorganism strain deposit center at 7.14 days 2022, with a deposit number of GDMCC 1.3533.
The LLRS-1 Strain described in "Can-Hua Lu, jun-Ying Li, meng-Ge Mi, et al complete Genome Sequence of Ralstonia syzygii subsp.indonesis Strain LLRS-1,Isolated from Wilted Tobacco in China.Phytopathology,2021,111:12:2392-2395" as "LLRS-1 of Ralstonia nectarii".
Ralstonia solanacearum (R.solanacearum) FQY _4 is a FQY _4 strain described in "Cao Yi, tian Baoyu, liu Yanxia, et al genome Sequencing of Ralstonia solanacearum FQY _4,Isolated from a Bacterial Wilt Nursery Used for Breeding Crop Resistance.Genome Announcements,2013,1 (3): e00125-13.
Solanaceae Ralstonia (R.solanacearum) CQPS-1 is a CQPS-1 strain described in "Liu Y, tang Y, qin X, et al genome Sequencing of Ralstonia solanacearum CQPS-1,a Phylotype I Strain Collected from a Highland Area with Continuous Cropping of Tobacco.Front.Microbiology,2017,8:974".
The pseudo Solanaceae Ralstonia (R.pseudosportanacrum) BSRS-1 and QBRS-1 are all strains stored in the laboratory of the applicant, who promises free release to the public within 20 years from the date of application of the present invention for verifying the effect of the present invention.
The culture medium and the culture seedling method adopted in the experimental example of the invention
1. Culture medium used
LB liquid medium contains 1% (w/v, the same applies below) tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose;
CG medium contained 0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone;
CGA contained 0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone, 1.5% agar;
TZC medium contains 1% peptone, 1% glucose, 0.1% casein hydrolysate, 1.5% agar and 0.005% triphenyltetrazolium chloride (TTC).
2. Floating seedling
Taking the Honghuadajinyuan of a disease-sensitive tobacco variety as a test object, and cultivating tobacco seedlings by floating seedling raising until the tobacco seedlings reach a 4-5 leaf period.
3. Pathogenic bacteria culture
Activating the surface of the Ralstonia RS on a TZC culture medium [1% peptone, 1% glucose, 0.1% casein hydrolysate, 1.5% agar and 0.005% triphenyltetrazolium chloride (TTC) ] by an ultralow temperature refrigerator at-80 ℃, and culturing in a constant temperature incubator at 28 ℃ for 36-48 h;
selecting a typical colony which has wider white edges, stronger mobility and pink or light red thin liquid in the middle, inoculating the typical colony into a triangular flask containing 100mL CG liquid culture medium (1% peptone, 1% glucose and 0.1% casein hydrolysate), and placing the triangular flask at 28 ℃ 225r/min for constant-temperature shaking culture for 24 hours;
diluting 100 μL to 10 -7 100 mu L of 10 -5 、10 -6 、10 -7 The diluted solution was spread on a TZC plate, and after 48 hours, the colony morphology was observed and the colony number was counted, and the amount of bacterial cells contained in the cultured bacterial solution was calculated.
Group 1 example, strain 18A6 of the present invention
The embodiment provides an agrobacterium (Agrobacterium deltaense) strain 18A6, which is characterized in that the preservation number is cctccc No: m2020041.
Any utilization, use, sale, offer for sale, production, preparation, culture, propagation and fermentation preservation number is CCTCC No: the behaviour of the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 falls within the scope of the invention.
According to the teaching and inspiring of the invention, for practical production, a person skilled in the art selects and prepares proper auxiliary materials by combining common technical means in the field of microbiological technology, and the preservation number of the invention is CCTCC No: the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 is formulated into various dosage forms, such as powder, tablet, liquid, etc., which meet the requirements of various technological processes.
The biocontrol bacteria, the agrobacterium 18A6, the strain 18A6, and the agrobacterium described in the summary and the detailed description refer to: the preservation number of the invention is CCTCC No: agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041.
Group 2 example, use of Strain 18A6 of the invention
The present set of embodiments provides a collection number of CCTCC No: use of agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 for controlling bacterial wilt.
In some embodiments, the bacterial wilt refers to tobacco bacterial wilt, a disease caused by infection by pseudomonas solanaceae ralstonia (Ralstonia solanacearum) or pseudomonas solanaceae ralstonia (Ralstonia pseudosolanacearum) or syzygote ralstonia (Ralstonia syzygii).
In other embodiments, agrobacterium (Agrobacterium deltaense) strain 18A6 does not control bacterial wilt by antagonizing or inhibiting solanacearum ralstonia (Ralstonia solanacearum) or pseudosolanacearum ralstonia (Ralstonia pseudosolanacearum) or syzygote ralstonia (Ralstonia syzygii).
Group 3 example, microbial inoculum of the invention
The present set of embodiments provides a microbial inoculum. All embodiments of this group share the following common features: the microbial inoculum comprises: the preservation number is CCTCC No: agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041.
In a further embodiment, the microbial agent further comprises: auxiliary materials.
In a more specific embodiment, the pharmaceutical excipients are selected from the group consisting of: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
In a specific embodiment, the dosage form is a powder.
The microbial inoculum is not limited to powder, and a person skilled in the art can select and blend proper auxiliary materials according to the teaching and inspiring of the invention and by combining common technical means in the field of microbial technology (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) for actual production, and the preservation number of the microbial inoculum is CCTCC No: the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 is formulated into various other dosage forms, such as tablets, liquids, sprays, granules, etc., which meet the requirements of various industrial processes.
Group 4 examples, methods of the invention for controlling bacterial wilt
The present set of embodiments provides a method of controlling bacterial wilt. All embodiments of this group share the following common features: the preservation number is CCTCC No: the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 prevents and treats bacterial wilt.
In a preferred embodiment, a bacterial concentration of 10 is used 7 -10 8 The pathogenic plants were root irrigated with a fermentation dilution of CFU/mL of agrobacterium (Agrobacterium deltaense) strain 18A6 or a suspension of bacterial powder at a bacterial concentration of 133.5 hundred million CFU/g.
In a specific embodiment, the diseased plant refers to: tobacco plants that develop bacterial wilt are infected with Ralstonia solanaceae (Ralstonia solanacearum) or Ralstonia pseudosolanaceae (Ralstonia pseudosolanacearum) or Ralstonia syringiensis (Ralstonia syzygii).
Experimental example 1, strain acquisition
1. Trapping, separating and culturing
Removing impurities and larger blocks from a soil sample, putting the soil sample into a glass culture dish with the diameter of 120mm, and wetting the soil with distilled water by adopting a titration bottle, wherein the soil layer is about 1.5cm thick;
preparation of a microorganism trapping device: firstly, coating glue on the edge of a microporous filter membrane with the diameter of 50mm and the aperture of 0.45 mu m, and placing a stainless steel flat-bottom gasket on the microporous filter membrane; 3mL of solid medium (1.2% gellan gum and 1.0% vitamins) was then added to the gasket lumen; coating the upper surface of the metal gasket with glue, and covering with another microporous filter membrane with the aperture of 0.45 μm;
placing the microorganism trapping device on the wet soil in the step (1), lightly compacting the device to ensure that the microporous filter membrane is fully contacted with the soil, then completely covering the device by using the residual soil, and wetting the soil again by using distilled water by using a titration bottle;
covering a culture dish, sealing the culture device with a sealing film, placing the culture device in a 30 ℃ incubator for culturing for 7d, observing the soil humidity during the culture, and supplementing with sterile water if the soil humidity is low;
taking out the culture device from the incubator, mashing the solid culture medium, adding 3mL of sterile water, standing for 10min, and gradient diluting to 10 -4 ；
Take 10 -4 、10 -5 The bacterial liquid is coated on an oligotrophic culture medium CN (containing 0.1% of casein amino acid, 0.1% of nutrient broth and 1.5% of agar), 5 dishes are coated on each gradient, dried on an ultra-clean workbench, and placed in a 30 ℃ incubator for 7d of culture;
2. experimental results
Through the experiment, picking from a CN culture dish, and scribing again on a CN culture medium to obtain pure bacteria 18A6; a single colony of 18A6 was picked up and inoculated into a test tube containing 2.5mL of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose), and subjected to constant temperature shaking culture at 28℃225r/min for 48 hours, the colony morphology of which is shown in FIG. 1. Experimental example 2, evaluation of control efficiency of biocontrol bacterium 18A6 in controlling tobacco bacterial wilt
The control efficiency evaluation is divided into indoor primary screening and secondary screening, and the specific operation is as follows:
step S1, raw measurement and preliminary screening
Treating tobacco seedlings: culturing tobacco seedlings by a floating seedling method to a 4-5 leaf period, taking out a standby tobacco Miao Lianggan floating tray from a seedling pool by 1d before seedling, making wounds on two sides of the root of the tobacco seedlings, which are 1.5cm away from the center of the tobacco plants, by using a sterile blade in the next day, and dividing the tobacco seedlings into a test group and a control group, wherein each group is 2 tobacco seedlings;
pretreatment of biocontrol bacteria: inoculating 1mL of test bacteria to the test group, and taking tobacco seedlings inoculated with LB culture medium as a control group;
culturing tobacco seedling root suspension: placing a thick plastic cloth with a size slightly larger than that of a floating tray for cultivating tobacco seedlings on a cultivation frame in a constant-temperature artificial climate chamber at 28 ℃, placing 5 disposable cultivation dishes at the 4 corners and the center of the plastic cloth as supports, placing the floating tray on the cultivation frame for cultivation for 1d, and watering by using a water spraying device three times in the morning, in the middle and in the evening, wherein the watering amount is suitable for preventing water in holes of the floating tray from dropping;
inoculation of pathogenic bacteria: after 1d of biocontrol bacteria pretreatment, 0.5mL of 10-time dilution of the Ralstonia RS is inoculated to a test group and a control group, and the culture is continued for 15 to 20d in a constant temperature artificial climate chamber at 28 ℃, and water is sprayed and moisturized by a water spraying device for three times in the morning, in the middle and in the evening;
observation record: and observing the morbidity conditions of the test group and the control group, recording the morbidity conditions of tobacco plants of the test group when the morbidity of the control group is more than 80%, recording the health of the tobacco plants as 1, the morbidity of the tobacco plants but not withered and assigned to 0.5, the withered and assigned to 0 of the tobacco plants, and selecting the highest-assigned strain in the tested bacteria as potential biocontrol bacteria for indoor rescreening, wherein the value of the tobacco plants treated by each tested bacteria is the sum of the assigned values of the two tobacco plants.
Step S2, biological test compound screen
Step S2, performing primary screening, wherein the rest operation steps are the same as those of the step S1 except for the following test.
1) The tested strain is potential biocontrol bacteria obtained by the primary screening of the biological detection in the step S1;
2) Increasing the number of the treated tobacco plants in the biological test re-screening, and treating 8 tobacco seedlings in both the treated group and the control group;
3) Disease occurrence of each treatment group strain was examined 10 and 20 days after inoculation with ralstonia, as shown in table 1.
TABLE 1 Effect of endophyte-proof 18A6 in growth chamber for preventing and treating tobacco bacterial wilt
Experimental results show that in the primary screening, two cigarettes treated by the biocontrol strain 18A6 are wilted and healthy, so that the value is 1.5; in the greenhouse rescreening, 7 and 5 tobacco plants were healthy at 10 and 20dpi, respectively, while all the control-treated tobacco plants had died. The result shows that 18A6 has better control effect and can be used for evaluating the control effect of greenhouse potted plants.
Experimental example 3, identification and preservation of Strain
1. Identification of strains
Conventional bacteria identification reference "Manual for identification of common bacterial systems" (edited by Dongxiu beads et al, science Press, 2001).
The Biolog GEN III plates were used to analyze the compound metabolic profile of biocontrol bacteria 18A6.
The molecular identification method comprises the following steps: the extraction kit of the bacterial genome DNA is prepared by the method which is described in the specification of the kit. The 16S rDNA sequence is amplified by a universal primer F27/R1492PCR, amplified under conventional conditions, amplified products are recovered by glue and then are connected with a vector pEAZY-T5 Zero vector, competent cells DH5 alpha of the escherichia coli are transformed by heat shock, colonies are picked up and colony PCR identification is carried out by taking M13F/M13R as a primer. The positive clones were sent to Shanghai Yingjun Biotechnology Co.Ltd for sequencing. The model species similar to biocontrol strain 18A6 were analyzed using the Ezbiocloud database (https:// www.ezbiocloud.net /), and the primary classification status of the genus strain was initially confirmed as shown in FIG. 2.
Further, genome sequencing technology is used to obtain the whole genome of the strain. And (3) comparing by using a TYPE database, calculating dDDH values of the species with the closest relationship between the patent strain and the strain, and finally determining the molecular classification status of the strain, as shown in figure 2.
And determining the classification status of the biocontrol bacteria by combining morphological and molecular biological characteristics.
The experimental results are recorded as follows:
1. culture characteristics and morphological characteristics:
the biocontrol bacteria 18A6 are cultured at the NA temperature of 28 ℃ to form smaller circular colonies after 36 hours, and the colonies are circular, semitransparent, milky and sticky after 48 hours. Bacterial gram-negative, rod-shaped, 0.46-0.60 μm wide, 0.62-1.45 μm long, with 2 to several flagella. The strain can grow at 20-37 deg.C and pH 5.0-10.0 and NaCl 0-3%.
3. Compound metabolic profile:
gen III shows that biocontrol bacteria 18A6 can utilize carbon sources including glucuronamide, acetoacetic acid, D-methyl lactate, L-arginine, L-lactic acid, bromo-succinic acid, pectin, N-acetyl-D-galactosamine, formic acid, methyl pyruvate, L-galacturonolactone, gamma-amino-butyric acid, alpha-D-glucose, alpha-D-lactose, D-sorbitol, beta-hydroxy-D, L-butyric acid, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-cellobiose, gentiobiose, L-rhamnose, D-fructose, glycerol, propionic acid, D-mannitol, D-maltose, stachyose, D-fructose, sucrose, D-mannose, D-melibiose, D-salicin, quinic acid, melezitose, D-trehalose, melibiose, beta-formyl-D-glucoside, L-fructose, N-acetyl-D-glucosamine, D-fructose-6-phosphate, inositol, D-malic acid, L-serine, L-alanine, L-histamine, L-aspartic acid, D-arabitol, D-galactose, D-glucose-6-phosphate, L-pyroglutamic acid, acetic acid, L-glutamic acid, L-malic acid, unavailable carbon sources are D-serine, p-hydroxy-phenylacetic acid, gelatin, alpha-hydroxy-butyric acid, N-acetylneuraminic acid, inosine, glyconic acid, citric acid, tween 40, mucic acid, alpha-keto-butyric acid, aminoacetyl-L-proline, 3-formylglucose, alpha-keto-glutaric acid, D-aspartic acid, N-acetyl-beta-D-mannosamine; the biocontrol bacterium 18A6 can grow under the conditions of guanidine hydrochloride, lithium chloride, pH 6, aztreonam, 1% sodium lactate, vancomycin, tetrazolium blue, potassium tellurite, lincolchicine, lincomycin SV, nalidixic acid, 1% NaCl, tetrazolium violet, and vinegar bamboo peach mycin, and cannot grow under the conditions of D-serine, 4% NaCl, 8% NaCl, pH 5, sodium butyrate, fusidic acid, minocycline, tetradec sodium sulfate, and sodium bromate.
3. Molecular identification of strains:
16S rDNA sequence analysis showed that biocontrol bacterium 18A6 and Agrobacterium deltaense YIC4121 in Agrobacterium T The highest similarity is 99.86%, and Rhizobium oryzihabitans M15 T Agrobacterium radiobacter (Agrobacterium radiobacter) ATCC 19358 T The sequence similarity of (2) was 99.79% and 99.57%, respectively. Analysis of the Type (stress) Genome Server (https:// types. Dsmz. De /) database revealed that biocontrol Strain 18A6 was found to be associated with A deltasense YIC4121 in Agrobacterium T The highest DNA molecular hybridization value (dDDH) is 81.6 percent (dDDH 4) which is more than 70.0 percent of the identification threshold value of the new species; next, A.leguminum MOPV5 T (66.9); the dDDH value of the biocontrol strain 18A6 and other agrobacterium is less than 60%. A phylogenetic tree is constructed based on the genome of the biocontrol bacterium 18A6 and the related species thereof, and the result shows that the biocontrol bacterium 18A6 and the agrobacterium (A.deltaense) standard strain YIC4121 T Gather into one branch (fig. 2). The above results indicate that biocontrol bacterium 18A6 is a.deltaense.
Table 2.18A6 comparative analysis of genomic similarity to closely related species
2. Preservation of Agrobacterium (A.deltaense) 18A6
From the above identification result, a strain of biocontrol strain 18A6 of Agrobacterium deltaense was confirmed and designated as 18A6, and the strain was sent to preservation with the following preservation information:
preservation number: cctccc NO: m2020041;
classification naming: agrobacterium deltaense 18A 18 6;
preservation date: 28 days of 1 month in 2020;
preservation unit: china center for type culture Collection;
preservation address: chinese, wuhan, university of Wuhan.
Experimental example 4, application of bacterial strain in prevention and treatment of tobacco bacterial wilt
Test setup: the method is carried out in a greenhouse with the temperature controlled between 28 and 30 ℃, and a Rolls' bacteria treatment group and a control group (the same volume of LB culture medium and tap water are irrigated) are arranged in the test, wherein the treatment is repeated for 3 times, and 10 tobacco plants are repeated each time;
test strain culture: culturing the biocontrol bacteria obtained by indoor screening with LB liquid culture medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose), and shake culturing at 30deg.C for 48 hr/min; laboratory-stored 6 strains of Ralstonia from Yuxi City (RS, LLRS 1), wenshan (QBRS 1), fujian (FQY _4) and Chongqing City (CQPS-1) were selected as pathogenic bacteria, and cultured with CG liquid medium (0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone) at 30deg.C for 24h at 225 r/min;
transplanting tobacco seedlings: tobacco seedlings cultivated by adopting a floating seedling cultivation mode are tobacco seedlings subjected to secondary leaf cutting, cultivation is carried out for about 50 days, red soil and organic matters are uniformly mixed during transplanting, and then the tobacco seedlings are transplanted;
inoculating: after transplanting, 250mL of biocontrol bacteria fermentation liquid is diluted by 25 times, 200mL of dilution liquid (18A 6 bacteria concentration is 10) is filled into each plant of tobacco 8 CFU/mL), the control group was treated with an equal volume of LB dilution; the CG culture medium was diluted 100-fold by shaking 24h of Rolls for the next day, and 200mL of Rolls was inoculated per strainThe TONGSHI bacterial dilution is inoculated by root irrigation, and the bacterial concentration of the Ralstonia bacterial dilution is 10 7 CFU/mL。
Investigation and statistics: the disease index was investigated every 7d after inoculation for 5 total surveys. The incidence rate, disease index and prevention and treatment effect of the tobacco bacterial wilt are calculated according to the following formula: incidence = diseased plants/total number of investigation plants x 100%; disease index = [ Σ (disease progression x number of strains of this grade)/(highest progression x total number of plants) ]x100; control effect= (control disease index-treatment disease index)/control disease index x 100%. The disease index is investigated according to the standard tobacco disease grading and investigation method of the tobacco industry of the people's republic of China (GB/T23222-2008), and the disease grading is as follows: the total plant has no disease of 0 level, the leaf withering of less than half of the disease side is 1 level, the leaf withering of less than half to two thirds of the disease side is 3 level, the leaf withering of more than two thirds of the disease side is 5 level, the leaf withering of the disease plant is 7 level, and the disease plant is basically withered to 9 level.
Test results: the biocontrol strain 18A6 is used for pretreating tobacco plants, and the next day is inoculated with the Ralstonia from different geographical sources, so that the occurrence of tobacco bacterial wilt can be effectively reduced. The biocontrol bacteria have prevention and control effects on bacterial wilt caused by 6 ralstonia bacteria from Yuxi City (RS, LLRS 1), wenshan (QBRS 1), lincang City (BSRS 1), fujian province (FQY _4) and Chongqing City (CQPS-1), and the prevention and control rate is 25.40-80.65%. Wherein, the biocontrol bacterium 18A6 has the best control effect on bacterial wilt caused by the Rolls Torili RS from the Yuxi city, which reaches 80.65 percent; secondly, the microbial inoculum is Ralstonia LLRS-1 from Yuxi city, and the control effect is 63.16%; the biocontrol bacterium 18A6 has better control effect on the Ralstonia BSRS-1 from the Lincang market, and the control effect is 52.54 percent. The bacterial wilt disease index of the tobacco strain treated by the biocontrol bacterium 18A6 is mostly lower than that of the control medicament thiabendazole, and the control effect of the biocontrol bacterium 18A6 is equivalent to that of the control medicament thiabendazole only when the ralstonia LLRS-1 from Yuxi city is inoculated. The results show that the biocontrol bacteria 18A6 have better control effect on bacterial wilt caused by the Ralstonia from different geographical sources, and the control effect is better than that of a control medicament, so that the biocontrol bacteria have certain development value (table 3).
TABLE 3 control Effect of biocontrol bacterium 18A6 on different Ralstonia
Experimental example 5, evaluation of greenhouse control efficiency of biocontrol microbial agent 18A6 on tobacco bacterial wilt
Experimental example 5 the procedure was the same as in experimental example 4, except for the following test and results.
1. Seed liquid culture: respectively picking a loop of biocontrol bacteria, inoculating into 500mL centrifuge tubes containing 500mL seed culture solution (peptone 10.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, agar 15.0g/L and pH 7.0), and placing into a shaking table at 28 ℃ for culturing for 15h.
2. Fermentation: the seed liquid of biocontrol bacteria is prepared by the steps of 1:100 proportion is transferred to a fermentation medium containing 150L of fermentation medium (5 g/L of glucose, 20g/L of bean pulp powder, 2.5g/L of bone peptone, 15g/L of corn starch, 2.5g/L of yeast extract, 0.5g/L of magnesium sulfate, 1g/L of dipotassium hydrogen phosphate and pH 7.3). The fermentation process parameters are as follows: (1) sterilization: the empty condition is: 0.15MPa,123 ℃ for 1h; the actual elimination conditions are as follows: 0.15MPa,123 ℃ for 35min; (2) inoculation: starting inoculation when the temperature of the sterilized culture solution is reduced to 37 ℃, wherein the inoculation ratio is 1:100 of the strain/culture solution; (3) fermentation control: the rotating speed is kept at 120r/min until the fermentation is finished; ventilation volume: hold for 15m 3 And/h, keeping until fermentation is finished; the tank pressure is kept at 0.05Mpa in the whole process; the ventilation is allowed to float for short periods of time for equipment reasons. Temperature: the whole fermentation process keeps the temperature at 37 ℃ and can float up and down by 0.5 ℃. Fermentation time: fermentation was run for 42h to give 241.8 hundred million CFU/mL broth.
3. Physical adsorption pulverization: centrifuging the fermentation liquor, collecting the centrifugal thalli as a raw material, mixing the raw material with diatomite according to a mass ratio of 1:1, adding sucrose accounting for 5%o of the total mass of the mixed material as a nutrient substance, airing the mixed material at a cool, dry and ventilated place, and crushing the mixed material when the moisture content reaches about 10%, thereby obtaining 18A6 powder, wherein the effective viable count is 133.5 hundred million CFU/g.
4. And (3) prevention and control: applying 2.5g of microbial inoculum to each tobacco seedling, adding 50mL of water for root irrigation, setting 3 times of repetition every 15 plants, and setting clear water as a control; the following day, the Rolls' bacteria QBRS-1 are inoculated, and each strain is 0.1OD; disease grade was investigated once a week and disease index was calculated.
Test results: as shown in FIG. 3, the disease of the control group was gradually increased with the increase of the inoculation days, while the disease of the tobacco strain treated by the microbial inoculum 18A6 was lighter, the area under the disease index curve was 36.68+ -19.91 at 24d after inoculation, and the disease index of the control group was 473.00 + -48.72. The control effect of the microbial inoculum 18A6 treatment group is 92.25% calculated by the area under the disease index curve, and the microbial inoculum has better control effect.
Experimental example 6 Effect of biocontrol bacterium 18A6 against Ralstonia
The bacteria inhibition zone of the strain is measured by adopting a flat plate counter culture method. The operation steps are as follows: CG medium (0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone) was gradient diluted to 10-4 for 24h; placing 100 μl of the diluted solution on the surface of CGA (0.1% acid hydrolyzed casein, 0.5% glucose, 2% peptone, 1.5% agar) culture medium, uniformly coating with the coated strain, and blow-drying under ultra-clean conditions; single colonies are selected and inoculated on the surface of a culture medium containing the Ralstonia, after 2-3 d of culture, the bacteriostasis condition of each strain is observed, the strain with the bacteriostasis effect is cultivated in a four-point method, and the bacteriostasis zone of the strain are measured (figure 4).
Test results: the results indicate that biocontrol bacterium 18A6 has no antagonistic ability to Ralstonia from Yuxi (RS and LLRS-1), lincang (BSRS-1) and Wenshan (QBRS-1), indicating that the biocontrol bacterium does not exert disease prevention effect in antagonism, and can exert effects by non-antagonistic niche competition, induced resistance and regulation of rhizosphere microecological structures.
| Ralstonia sp | Antibacterial circle/cm | Antibacterial zone/cm |
| RS | 0.00±0.00 | 0.00±0.00 |
| LLRS-1 | 0.00±0.00 | 0.00±0.00 |
| BSRS-1 | 0.00±0.00 | 0.00±0.00 |
| QBRS-1 | 0.00±0.00 | 0.00±0.00 |
Claims (10)
1. An agrobacterium (Agrobacterium deltaense) strain 18A6, characterized by a collection number of CCTCC No: m2020041.
2. The preservation number is CCTCC No: use of agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 for controlling bacterial wilt.
3. The preservation number of claim 2 is CCTCC No: use of agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 for controlling bacterial wilt, wherein the bacterial wilt is a tobacco bacterial wilt, a disease caused by infection with pseudomonas solanacearum (Ralstonia solanacearum) or pseudomonas solanacearum (Ralstonia pseudosolanacearum) or catwalk typhi (Ralstonia syzygii).
4. A preservation number according to claim 2 or 3 is CCTCC No: use of agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 for controlling bacterial wilt, characterized in that agrobacterium (Agrobacterium deltaense) strain 18A6 does not control bacterial wilt by antagonizing or inhibiting solanacearum rogowski (Ralstonia solanacearum) or pseudosolanacearum rogowski (Ralstonia pseudosolanacearum) or cattail peach rogowski (Ralstonia syzygii).
5. A microbial inoculum, characterized by comprising: the preservation number is CCTCC No: agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041.
6. The microbial agent of claim 5, further comprising: auxiliary materials.
7. A bacterial agent according to claim 5 or 6, wherein the dosage form is selected from: fermentation broth, powder and suspension emulsion.
8. A method for preventing and treating bacterial wilt is characterized in that the preservation number is CCTCC No: the agrobacterium (Agrobacterium deltaense) strain 18A6 of M2020041 prevents and treats bacterial wilt.
9. The method for preventing and treating bacterial wilt according to claim 8, wherein the bacterial concentration is 10 7 -10 9 The pathogenic plants were root irrigated with a fermentation dilution of CFU/mL of agrobacterium (Agrobacterium deltaense) strain 18A6 or a suspension of bacterial powder at a bacterial concentration of 133.5 hundred million CFU/g.
10. A method for controlling bacterial wilt according to claim 8 or 9, wherein said diseased plant is: tobacco plants that develop bacterial wilt are infected with Ralstonia solanaceae (Ralstonia solanacearum) or Ralstonia pseudosolanaceae (Ralstonia pseudosolanacearum) or Ralstonia syringiensis (Ralstonia syzygii).
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