CN117546872A - Prevention and control method for sclerotinia rot of sunflower - Google Patents
Prevention and control method for sclerotinia rot of sunflower Download PDFInfo
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- CN117546872A CN117546872A CN202311559622.3A CN202311559622A CN117546872A CN 117546872 A CN117546872 A CN 117546872A CN 202311559622 A CN202311559622 A CN 202311559622A CN 117546872 A CN117546872 A CN 117546872A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention relates to the technical field of microorganisms and application thereof, in particular to a method for preventing and controlling sclerotinia rot of sunflower discs; culturing a small-volume biocontrol bacterial liquid, fermenting the biocontrol bacterial liquid, and mixing the two biocontrol bacterial liquids to obtain a composite biocontrol bacterial liquid and applying the composite biocontrol bacterial liquid to realize the prevention and control of the sunflower disc rot sclerotium disease; the application of the composite biocontrol bacteria liquid can be realized by spraying the composite biocontrol bacteria on the direct flower discs and spreading the biocontrol bacteria carried by bees to the sunflower flower discs by spraying the composite biocontrol bacteria on bees, so that the efficient prevention and control of the sunflower disc rot type sclerotinia rot can be realized.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to a method for preventing and controlling sclerotinia rot of sunflower.
Background
Sclerotinia sclerotiorum of sunflower is a fungus disease caused by sclerotinia sclerotiorum, and the pathogenic bacteria are sclerotinia sclerotiorumSclerotinia sclerotiorumThe pathogenic mechanism is mainly that the parasitic plant cell wall is degraded by secreting hydrolytic enzymes and oxalic acid toxin is generated to destroy host cell tissues, pathogenic bacteria exist in the forms of sclerotium, ascospores and mycelium, and the pathogenic bacteria can infect from different parts of sunflower roots, stems, leaves, flower discs and the like to cause root rot, stem rot, leaf rot and disc rot, and when the disease is serious, the whole sunflower plant death is caused, the propagation speed is high, the range is wide, and the large-area yield reduction or the sterilization is caused. Only the average annual yield reduction of the inner Mongolia planting area is over 20 percent, the average annual economic loss reaches about 30 hundred million yuan, and the average annual economic loss nationwide reaches the scale of 50 hundred million yuan.
The sclerotium bacteria prefer a low-temperature high-humidity environment, ascospores released by ascospores formed by sclerotium germination (figure 5) after rainfall in the sunflower flowering period are ejected and scattered on the sunflower disc, so that the sunflower disc rot is caused (figure 4), the disease is extremely difficult to control, and huge economic loss is brought to oil crop production including sunflowers.
For preventing and controlling the sclerotium disease of sunflower, generally, field cultivation management, disease-resistant variety selection and medicament prevention and control methods are adopted, so that a great amount of cost is increased, and the method has very limited effect on preventing and controlling the sclerotium disease of sunflower; meanwhile, the plants Gao Dayu are usually closed in the later growth period of the sunflower, the manual pesticide spraying in the field is difficult, if the unmanned aerial vehicle is used for pesticide spraying, because the later sunflower flower disc sags, the front face of the flower disc faces downwards, and the unmanned aerial vehicle cannot effectively spray the pesticide from the upper face to the front face of the flower disc, so that the prevention and control effect is not ideal; in addition, long-term use of the drug for prevention and treatment can also cause mutation of pathogenic bacteria, promote drug resistance and generate new drug resistance to weaken the prevention effect of the chemical agent; too much application of chemical pesticides can also seriously affect the quality of sunflower seeds and edible safety; therefore, research and development of an effective prevention and control method for sunflower sclerotium disease is a potential method for overcoming the current situation that the disease-resistant variety is insufficient and the overreliance on chemical pesticides is avoided.
Disclosure of Invention
The invention provides a method for preventing and controlling sclerotium disease of sunflower, and aims to provide a method for preventing and controlling the specific preventing and controlling effect of sclerotium disease of sunflower.
The technical scheme adopted by the invention is as follows: a method for preventing and controlling sclerotinia rot of sunflower comprises the following steps:
step 1, culturing a small-volume biocontrol bacterial liquid to obtain bacillus subtilisBacillus subtilisSmall-volume biocontrol bacterial liquid of KB3 and bacillus megatheriumBacillus megateriumTZ-1 is a small-volume biocontrol bacterial liquid;
step 2, fermenting the biocontrol bacterial liquid to obtain bacillus subtilisBacillus subtilisKB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megateriumA biocontrol bacterial liquid of TZ-1;
step 3, mixing two biocontrol bacteria liquid to obtain a composite biocontrol bacteria liquid;
and step 4, applying the composite biocontrol bacterial liquid.
Further, the step 1 specifically includes the following steps:
s101, preparing bacillus subtilisBacillus subtilisKB3 and Bacillus megateriumBacillus megateriumTZ-1, respectively inoculating two strains of biocontrol bacteria to an LB culture medium for culture;
s102, selecting single bacterial colonies, preparing bacterial cakes, and respectively obtaining bacillus subtilisBacillus subtilisKB3 bacterial cake and bacillus megatheriumBacillus megateriumA cake of TZ-1;
s103, respectively transferring the bacterial cakes obtained in the step S102 to a trayShake culturing in 100mL liquid LB culture medium container at 28deg.C under shaking culture conditions of 180rpm to obtain Bacillus subtilisBacillus subtilisSmall-volume biocontrol bacterial liquid of KB3 and bacillus megatheriumBacillus megateriumTZ-1 is a small-volume biocontrol bacterial liquid.
Further, the step 2 specifically includes the following steps:
s201, preparing 7-8L of liquid LB culture medium, and adding the liquid LB culture medium into a fermentation tank;
s202, introducing water vapor into the interlayer of the fermentation tank, and externally heating to 80 ℃;
s203, when the temperature of the interlayer reaches 80 ℃, opening a valve for allowing water vapor to enter the fermentation tank, introducing the water vapor into the fermentation tank, heating the culture medium to 115 ℃, and sterilizing for 20min;
s204, stopping introducing water vapor, introducing air, cooling the tank body and the steam filter element, discharging redundant water vapor out of the fermentation tank, and automatically injecting cold water into an interlayer of the fermentation tank to gradually cool the culture medium;
s205, when the temperature is reduced to 28 ℃, opening a fermentation tank, igniting absorbent cotton with alcohol absorbed in an inoculating loop at the opening of the fermentation tank, adding 250ml of the biocontrol bacterium liquid obtained in the step 1 under the flame sterile condition, keeping the working state of an air compressor, opening a switch for intermittent stirring of the fermentation tank under the condition of ensuring continuous ventilation to the fermentation tank, and after the fermentation tank is closed for more than 24 hours, respectively obtaining bacillus subtilis after the bacterium liquid grows into a slightly turbid stateBacillus subtilisKB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megateriumTZ-1 biological control bacterial liquid.
Further, the step S205 specifically includes the following steps: the intermittent stirring is specifically carried out for 2min, and the alternating circulation stirring is stopped for 5 min.
Further, the step 3 specifically comprises the following steps of mixing the two biocontrol bacteria liquid obtained in the step 2 in equal proportion to obtain a composite biocontrol bacteria liquid, and configuring the composite biocontrol bacteria liquid according to the concentration of 1X 108 CFU/mL.
Further, after 2d of the flowering period of the sunflower and during the full-bloom period, the composite biocontrol bacterial liquid obtained in the step 3 and water are mixed and diluted according to the proportion of 1:9, and are uniformly sprayed on the surface of the flower disc of the sunflower.
Further, the sunflower is uniformly sprayed on the surface of the flower disc of the sunflower through a spraying device, and the sunflower is planted according to the wide and narrow rows: the wide row is 190m, the narrow row is 60cm, and the spraying device is used for driving between the wide rows.
Further, the step 4 specifically comprises the following steps of obtaining the composite biocontrol bacterial liquid in the step 3 according to the composite biocontrol bacterial liquid: the water is 1:10 are mixed and diluted in proportion and are sprayed at the inlet and outlet of beehive bees.
The method for preventing and controlling sclerotium disease of sunflower head according to claim 8, wherein 10mL of bacterial liquid is sprayed in each beehive every 2 hours.
The beneficial effects achieved by the invention are as follows:
1. the invention realizes the efficient prevention and control of the sunflower disc rot type sclerotium disease by directly spraying the flower disc and spraying the flower disc onto bees to enable the bees to carry biocontrol bacteria to spread to the sunflower disc.
2. The bee passes bacteria, has extremely strong pertinence, and brings biocontrol bacteria to each pistil when the bees collect honey, and prevents and treats the bees point by point.
3. The use of biocontrol bacteria reduces the application amount of pesticides, reduces the cost and simultaneously reduces the phytotoxicity.
Drawings
FIG. 1 is a schematic diagram showing the antagonistic ability of two biocontrol strains of the invention on a plate.
FIG. 2 is a schematic illustration of a plate crossing experiment of the present invention.
FIG. 3 is a flowchart of the method for controlling sclerotium disease in sunflower according to the present invention.
Fig. 4 is a schematic representation of sunflower disc rot of the invention.
FIG. 5 is a schematic representation of a sclerotinia sclerotiorum ascalonicum in a field.
FIG. 6 is a schematic diagram of a composite biocontrol bacterial liquid according to the invention.
FIG. 7 is a schematic diagram of a composite biocontrol bacterial liquid according to the invention.
Fig. 8 is a schematic diagram of a field spray composite biocontrol bacteria liquid.
Fig. 9 is a graph of nectar collected by bees carrying the composite biocontrol bacteria liquid.
Fig. 10 is a schematic diagram of a hive spray composite biocontrol bacteria liquid.
Detailed Description
The background of the present application will be described first: whether the biological control of the sclerotium disease of the sunflower is successful or not is critical to whether the high-efficiency biocontrol strain can be screened, and the high-efficiency biocontrol strain is separated and screened from a large amount of sunflower seeds and pasture materials by constructing a high-efficiency biocontrol strain screening system, and bacillus subtilisBacillus subtilisKB3, accession number: CGMCC No.23108 and Bacillus megateriumBacillus megateriumTZ-1 with the preservation number of CGMCCNO.22296.
Bacillus subtilisBacillus subtilisKB3 was isolated from sunflower seeds of the JK601 variety collected from the plant disease laboratory at the inner Mongolian agricultural university, 4 th year 2021, and this strain was designated: KB3 and preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 8 th month 3 of 2021, wherein the preservation address is the national academy of sciences of China, which is North Chen Xiyu No. 1, the Korean region, and the classification and naming:Bacillus subtilisthe preservation number is: CGMCC No.23108.
Bacillus megateriumBacillus megateriumTZ-1 was isolated from: alfalfa experimentally collected from the new district of the university of inner mongolia agriculture at 5 months 2020: the bacterium is named as: TZ-1 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 5 months and 10 days in 2021, wherein the preservation address is the national academy of sciences of China, which is North Chen West Lu No. 1, and the Beijing is the Chapter of Korea, and the classification and naming are that:Bacillus megateriumthe preservation number is: CGMCC No.22296.
Measuring antagonistic capacity of two biocontrol strains on a flat plate by using a sclerotinia sclerotiorum target; as shown in FIG. 1, bacillus subtilis was found in the bacteriostatic activity experimentBacillus subtilisKB3 strain can inhibit the growth of sclerotinia sclerotiorum mycelium, but bacillus megateriumBacillus megateriumTZ-1 strain had no inhibitory effect on sclerotinia mycelium, and the results are shown in Table 1 below:
for bacillus subtilisBacillus subtilisKB3 and Bacillus megateriumBacillus megateriumTZ-1 plate crossover experiments were performed: the results of the plate crossing experiments show that no mutual inhibition exists between the two effective biocontrol strains, and the two effective biocontrol strains can be used as original strains for preparing the microorganism composite fermentation broth, and the experimental results are shown in figure 2.
As shown in fig. 3, the invention provides a method for preventing and controlling sclerotinia rot of sunflower, which comprises the following steps:
step 1, culturing a small-volume biocontrol bacterial liquid;
s101, using a plate scribing method to treat bacillus subtilisBacillus subtilisKB3 and Bacillus megateriumBacillus megateriumTZ-1, two strains of biocontrol bacteria are respectively inoculated to LB culture medium; culturing in a 28 ℃ incubator under dark condition for 2d;
s102, selecting healthy and sterile single colonies, and using a sterilized puncher to perform bacterial cake to respectively obtain bacillus subtilisBacillus subtilisKB3 bacterial cake and bacillus megatheriumBacillus megateriumA cake of TZ-1;
s103, respectively transferring the bacterial cakes obtained in the step S102 into triangular flasks filled with 100mL of liquid LB culture medium, and shake-culturing for 12h at 28 ℃ under a shaking table of 180rpm to obtain bacillus subtilisBacillus subtilisSmall-volume biocontrol bacterial liquid of KB3 and bacillus megatheriumBacillus megateriumTZ-1 is a small-volume biocontrol bacterial liquid.
Step 2, fermenting biocontrol bacteria liquid;
s201, cleaning and disinfecting the interior of a fermentation tank (fermentation tank, shanghai Baoxing biological equipment engineering Co., ltd., stainless steel fermentation tank, model: biotech-JS/JSA), then preparing 7-8L of liquid LB culture medium, and adding the liquid LB culture medium into the fermentation tank;
s202, adding distilled water into a distilled water bucket (a steam engine, shanghai Huazhi special boiler manufacturing Co., ltd., model: HX-7.5D) of a steam engine, starting up and heating to evaporate distilled water into steam, opening a steam valve of a fermentation tank after steam extraction to enable the steam to be introduced into an interlayer of the fermentation tank, and externally heating to 80 ℃;
s203, when the temperature of the interlayer reaches 80 ℃, opening a valve for allowing water vapor to enter the fermentation tank, introducing the water vapor into the fermentation tank, heating the culture medium to 115 ℃, and sterilizing for 20min;
s204, after sterilization, closing a steam engine and a steam valve to stop introducing steam, opening an air compressor, an air inlet valve and an air outlet valve (air compressor, shanghai Huai Xin electromechanical equipment limited company, model: PA 98-1), introducing air, cooling a tank body and a steam filter element, discharging redundant steam out of a fermentation tank, and automatically injecting cold water into an interlayer of the fermentation tank to gradually cool a culture medium;
s205, when the temperature is reduced to 28 ℃, opening a fermentation tank, igniting absorbent cotton which is sucked with alcohol and is in an inoculating loop at the opening of the fermentation tank, adding 250ml of one biocontrol bacterium liquid obtained in the step 1 under the flame sterile condition, keeping the working state of an air compressor, opening a switch for intermittently stirring the fermentation tank under the condition of continuously ventilating the fermentation tank, keeping the operation for 2min, stopping 5min of alternate circulation stirring, sealing and fermenting for more than 24h, taking out the fermented biocontrol bacterium liquid from the fermentation tank, sealing for standby, and the other biocontrol bacterium fermentation mode is the same; respectively obtaining bacillus subtilisBacillus subtilisKB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megateriumTZ-1 biological control bacterial liquid.
Step 3, mixing two biocontrol bacteria solutions;
mixing the two biocontrol bacteria liquid obtained in the step 2 in equal proportion to obtain a composite biocontrol bacteria liquid, and configuring the composite biocontrol bacteria liquid according to the concentration of 1X 108 CFU/mL; the obtained composite biocontrol bacteria liquid is shown in fig. 6-7.
Bacillus megateriumBacillus megateriumTZ-1 biocontrol bacteria liquid characteristics: no pungent smell, milky yellow color, slightly turbid bacterial solution.
Bacillus subtilisBacillus subtilisKB3 biocontrol bacteria liquid characteristics: has slight indole taste, yellow brown color and slightly turbid bacterial liquid.
Composite biocontrol bacteria liquid: each liter of the bacterial liquid is 1000ml of water, 10g of peptone, 10g of sodium chloride and 5g of yeast powder, and the substances produced by the bacterial cells.
Step 4, applying the composite biocontrol bacterial liquid;
the method comprises the following steps: and (3) after the sunflower blooming period is 2d and the full bloom period, mixing and diluting the composite biocontrol bacterial liquid obtained in the step (3) with water according to the proportion of 1:9, and uniformly spraying the composite biocontrol bacterial liquid on the surface of the sunflower discs through a spraying device (sprayer and pesticide spraying machine).
The reason for applying the drug in the full bloom period after 2d of the sunflower is as follows: the ascomycetes of the pathogenic bacteria are discovered for the first time from the beginning of 8 months to the beginning of 9 months, and the ascospores continuously germinate and scatter to infect, so that biocontrol bacteria are required to be sprayed to sunflower discs for multiple times to protect; the two spraying times are carried out at an interval of one week, and the more early release and less later release of the ascus disk are mainly considered; and if the early-stage biocontrol bacteria are propagated on the flower disc in a large quantity by utilizing the nutrition of chemical drugs to form a protective layer, the ascospores are prevented from infecting the flower disc.
The reason for the surface of the flower disc of the sunflower with the composite biocontrol bacteria liquid is as follows: the sclerotium disease of sunflower disc rot is ascospores formed by germination of sclerotium in soil, spread in air, spread to sunflower plants along with airflow, and spread on sunflower discs, so that the ascospores are easy to infect from the front of the sunflower discs to cause the disc rot, thereby forming disc rot; the composite biocontrol bacterial liquid is sprayed on the front surface of the sunflower disc, so that the bacterial liquid covers the surface of the sunflower disc, and the invasion of ascospores to the sunflower disc can be accurately blocked (the front and the back of the sunflower disc can be sprayed, and the effect of comprehensive prevention and control can be achieved).
Wherein, sunflower is planted according to wide and narrow rows: a pesticide spraying machine can be driven between the wide rows of 190m and the narrow rows of 60 cm;
the pesticide spraying machine is an independent refitted pesticide spraying machine, the machine body is a small crawler tractor, the front of the water tank device is provided with 5 spray heads on each side from high to low on two sides of the rear of the machine body, and the spraying height can be freely adjusted. The design can uniformly and comprehensively spray from different positions of the sunflower disc height, the blade height and the ground surface, and each spray head can manually adjust a switch according to requirements so as to achieve the purpose of specific spraying at specific positions; the maximum capacity of the water tank of the pesticide spraying machine is 250L, 200L of liquid is added in each working, the whole field area is 5 mu, and 200L of liquid can be sprayed in one working; the sprayer is a knapsack electric sprayer with a capacity of 15L.
The second method is as follows: and (3) obtaining the composite biocontrol bacterial liquid in the step (3), and mixing the composite biocontrol bacterial liquid with the composite biocontrol bacterial liquid: the water is 1:10, 50mL is taken and put into a watering can, 10mL of bacterial liquid is sprayed on each beehive every 2h in the time period of honey collection of bees in the daytime at the inlet and outlet of the beehive, when bees collect honey in the beehive, biocontrol bacterial liquid is dipped at the inlet of the beehive, and when honey is collected, bees bring the bacterial liquid to each floret on each sunflower disc.
Adopts the principle of preventing and controlling sclerotinia by biocontrol bacteria and bees: the bee can be used as the nectar of the sunflower in the flowering phase of the sunflower, and the bee is fully contacted with each tubular flower on the sunflower disc in the collecting process, so that the close contact between the bee and the disc is considered to have important effects on the occurrence and development of the sunflower disc rot and the prevention and control of the disease through long-time observation; in other words, the bee is a double-edged sword which can not only carry pathogenic bacteria to spread on each small flower to cause the disc rot, but also carry biocontrol bacteria to each tubular flower of the flower disc to prevent and treat the disc rot; in order to prevent bees from spreading pathogenic bacteria, biocontrol bacteria are sprayed on bees, on one hand, the biocontrol bacteria are considered to kill pathogenic bacteria harmful to sunflowers carried on the bees, and on the other hand, the biocontrol bacteria are transferred to sunflower discs by the bees, so that the biocontrol bacteria inhibit the pathogenic bacteria on the flower discs to protect the flower discs from being infected by the pathogenic bacteria; this approach has two advantages: the honey collection mode of the precise bacteria-applying bees is very precise, and each floret is hardly omitted, so that if the biocontrol bacteria are sprayed on the bees, the biocontrol bacteria are precisely applied, and the method avoids using manpower spraying and mechanical spraying, so that the time can be saved, and the cost of mechanical spraying can be saved.
Experiment one, indoor composite biocontrol bacteria liquid effect experiment on sunflower sclerotinia
Preparing a sunflower sclerotinia prevention effect test by using a pot culture, wherein 8 pots are respectively arranged in an experimental group and a control group; filling field soil into pot with 20cm multiplied by 20cm, wherein the distance between the soil surface and the pot edge is 5cm, adding 20g of sclerotinia bacteria powder to each pot soil surface of an experimental group and a control group, mixing the soil, and covering the soil for 2cm; sowing sunflower seeds of the sunflower variety LD5009 after preparing the disease soil of the experimental group and the control group for two days; adding the composite biocontrol bacteria liquid (about 1 multiplied by 108 cfu/mL) into the experimental group, and respectively adding water with the same volume into the control group; investigation and statistics of the disease condition of sunflower plants after 35d sowing; the 3 replicates were set, the disease grade of sunflower plants was observed and recorded, and the disease index was calculated (table 1).
As shown in table 2 below, the results of test one are as follows: after being treated by the compound biocontrol bacteria liquid, the roots and stems of the sunflowers have no obvious disease symptoms, and the roots and stem bases of the sunflowers in the control group have typical rot symptoms: the disease index is 9.84 respectively, and compared with the control group, the disease index is reduced by 47.1: the control effect of the composite biocontrol bacterial liquid on the sclerotinia sclerotiorum of sunflower in seedling stage is 82.72 percent.
Test two, prevention and treatment effects of single plant biocontrol bacterial liquid on sclerotinia sclerotiorum in sunflower seedling stage
Two experimental components of the second experiment were respectively added with bacillus subtilisBacillus subtilisKB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megateriumA biocontrol bacterial liquid of TZ-1; the remaining test conditions were the same as those of test one.
As shown in the following Table 3, the second test results are as follows, and the results show that the rootstalk of sunflower after being treated by the single plant biocontrol bacterial liquid has no obvious disease symptoms, and the results show that the bacillus megatheriumBacillus megateriumTZ-1, bacillus subtilisBacillus subtilisThe disease index of the KB3 treatment group is respectively 12.62 and 11.69, compared with a pair of photographs (without biocontrol bacteria liquid addition) in the test, the disease index is respectively 44.32 and 45.25,Bacillus megateriumbiological control bacterial liquid of TZ-1Bacillus subtilisThe control effect of the biocontrol bacterial liquid of KB3 on the sclerotinia sclerotiorum of sunflower is 77.85 percent and 79.47 percent respectively.
The combination test I and the test II show that the control effect of the composite fermentation broth on the sclerotinia sclerotiorum of sunflower in the seedling stage is 82.72 percent (table 2),Bacillus megateriumbiological control bacterial liquid of TZ-1Bacillus subtilisThe biocontrol bacterial liquid of KB3 has the control effects on seedling stage sunflower sclerotinia disease of 77.85 percent and 79.47 percent (Table 3), so that the control effect on diseases is more obvious by applying the composite fermentation bacterial liquid than by applying the single biocontrol bacterial liquid.
After screening two strains of biocontrol bacteria, analyzing whether the biocontrol bacteria can directly antagonize the pathogenic bacteria, and analyzing to find that KB3 has antagonistic capability, butBacillus megateriumTZ-1 does not have any antagonistic capacity; the strain with antagonistic capability mainly inhibits pathogenic bacteria directly, and the strain without antagonistic capability does not inhibit the disease indirectly by directly inhibiting bacteria, possibly improving the resistance to pathogenic bacteria by inducing sunflower; the two functional bacteria with different disease prevention strategies are combined for use, so that pathogenic bacteria can be directly inhibited, and sunflower disease resistance can be induced to prevent and treat diseases at multiple angles; the effect of preventing diseases by compounding two biocontrol bacteria is better than that of single bacteria, which proves that the synergistic function of compounding the two biocontrol bacteria on preventing and treating sclerotinia sclerotiorum.
Outdoor field test of sunflower treated by composite biocontrol bacteria liquid
Introducing outdoor field environment, sowing addresses: the Wulan Bo-Hu market is observed Ha Eryou Yinqian rose town Ha Lagou village, and the 6-year sunflower sclerotium disease high incidence test field is continued;
sowing date: 2023, 5, 20;
sowing area: the total area of the common sowing sunflowers is 5 mu;
cultivation mode: wide and narrow row planting: a pesticide spraying machine can be driven between the wide rows of 190m and the narrow rows of 60 cm;
plant spacing and row spacing for sowing: the plant spacing is 55cm, the row spacing is 190cm for each wide row, 60cm for each narrow row, and the sowing is performed in a wide-narrow alternating mode;
number of plants sown per mu: 1200 plants are sown per mu.
In the first experimental group, 10L of the composite biocontrol bacteria liquid and 190L of water are mixed and diluted, and are added into a pesticide spraying machine, and 40L of the composite biocontrol bacteria liquid is sprayed per mu; dividing 10L of bacterial liquid into 20 parts, adding each part of bacterial liquid and 9.5L of water into sprayers, preparing 10L of bacterial liquid, and spraying each sprayer for 0.25 mu;
as shown in fig. 8, the flowering period of sunflower is 8 months old, 2d after flowering (8 months and 7 days), and the full-bloom period (8 months and 14 days), and the two biocontrol bacteria are uniformly sprayed on the surface of the flower disc of sunflower after being mixed in equal proportion;
most sunflowers in the field already bloom in 8 months and 7 days, the flowering degree of the flower disc is about 50%, the sunflower flower disc is in a sun-facing state, and the right side of the flower disc faces upwards; most sunflower discs in the field in 8 months and 14 days are all flowering, and most flower discs face downwards. The ascus discs are found in the field for the first time from 8 months in 2023 to 3 days, the ascus discs germinate in a large amount due to rainfall between the field soil and the soil from 8 months to 6 days, and bacterial liquid is sprayed twice after the ascus discs are found.
In the second experimental group, the composite biocontrol bacteria liquid is sprayed to bees at the edge of a sunflower field, the bees carry biocontrol bacteria to inoculate on a flower disc, and the composite biocontrol bacteria liquid is not directly sprayed on the flower disc;
seeding address: sunflower test fields in the test bases of the Ulan Botryn institute of agriculture and forestry science;
sowing date: 2023, 5, 18;
sowing area: the total area of the common sowing sunflowers is 5 mu;
plant spacing and row spacing for sowing: the plant spacing is 55cm, the row spacing is 190cm for each wide row, 60cm for each narrow row, and the sowing is performed in a wide-narrow alternating mode;
number of plants sown per mu: 1200 plants are sown per mu;
the composite biocontrol bacterial liquid is prepared by the following steps: the water is 1:10, mixing and diluting in proportion, and filling 50ml of the mixture into a watering can; as shown in fig. 10, 10mL of bacterial liquid is sprayed on each beehive every 2 hours at the entrance and exit of beehives, 8:00 to 16 pm: 00 is sprayed for 5 times; when bees go out to collect honey, biological control bacterial liquid is dipped at the inlet of the beehive; as shown in FIG. 9, when nectar is collected, bees bring the bacterial liquid to each floret on each sunflower disc.
A control group I, wherein chemical agents are sprayed for prevention and control;
the cultivation mode, the seeding variety and the mu seeding number of the control field are the same as those of the test field;
medicament: pyraclostrobin emulsifiable concentrate;
spraying concentration: 300 g/mu;
spraying time: day 7, day 28, day 8, and day 8;
the spraying method comprises the following steps: the pyraclostrobin emulsifiable concentrate is diluted with 300g per mu and 1500 times of water, 450L of medicament is prepared and added into a pesticide spraying machine, and 9L of the pesticide is sprayed per mu.
A second control group, a farmer mode;
seeding address: sowing 300 mu of sunflowers in farmlands near the Hara ditch villages in the rose towns;
sowing date: 2023, 5, 25;
sowing varieties: the same glow 15;
sowing modes: row spacing: 65cm plants with a spacing of 60cm and 1800 plants per mu;
spraying 500 times of 40% dimethachlon wettable powder in the full bloom stage; the spraying is carried out 2 times continuously every 7 days for 1 time.
The results of the experiments are shown in table 4 below,
in the first test group, the incidence rate of sclerotium disease of the disc rot type fungus, which is subjected to the biocontrol fungus spraying treatment of the flower disc during harvesting, is 0; the incidence rate of the disc rot of the experiment group two bacteria and bees combined cell experiment is 0.76%; the first control group is to spray chemical agent only for prevention and treatment, and the incidence rate of the disc rot is 4.58%; in the second control group, the new-crop sunflower is planted in a mode of farmers nearby the test field, the planting area is about 300 mu, and the incidence rate of the disc rot reaches 20%; therefore, the biological control bacteria spraying or bacteria and bee combination measures are adopted, and the biological control bacteria spraying or bacteria and bee combination measures have ideal control effects.
The embodiments of the present invention described above do not limit the scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention as set forth in the appended claims.
Claims (10)
1. The method for preventing and controlling the sclerotium disease of sunflower is characterized by comprising the following steps:
step 1, culturing a small-volume biocontrol bacterial liquid to obtain bacillus subtilisBacillus subtilis Small-volume biocontrol bacterial liquid of KB3 and bacillus megatheriumBacillus megaterium TZ-1 is a small-volume biocontrol bacterial liquid;
step 2, fermenting the biocontrol bacterial liquid to obtain bacillus subtilisBacillus subtilis KB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megaterium A biocontrol bacterial liquid of TZ-1;
step 3, mixing two biocontrol bacteria liquid to obtain a composite biocontrol bacteria liquid;
and step 4, applying the composite biocontrol bacterial liquid.
2. The method for preventing and controlling sclerotium disease of sunflower head according to claim 1, wherein the step 1 specifically comprises the following steps:
s101, preparing bacillus subtilisBacillus subtilis KB3 and Bacillus megateriumBacillus megaterium TZ-1, respectively inoculating two strains of biocontrol bacteria to an LB culture medium for culture;
s102, selecting single bacterial colonies, preparing bacterial cakes, and respectively obtaining bacillus subtilisBacillus subtilis KB3 bacterial cake and bacillus megatheriumBacillus megaterium A cake of TZ-1;
s103, respectively transferring the bacterial cakes obtained in the step S102 into a container filled with 100mL of liquid LB culture medium, and shake-culturing for 12 hours under the shaking culture condition of a shaking table at 28 ℃ and 180rpm to obtain bacillus subtilisBacillus subtilis Small-volume biocontrol bacterial liquid of KB3 and bacillus megatheriumBacillus megaterium TZ-1 is a small-volume biocontrol bacterial liquid.
3. The method for preventing and controlling sclerotium disease of sunflower head according to claim 1, wherein the step 2 specifically comprises the following steps:
s201, preparing 7-8L of liquid LB culture medium, and adding the liquid LB culture medium into a fermentation tank;
s202, introducing water vapor into the interlayer of the fermentation tank, and externally heating to 80 ℃;
s203, when the temperature of the interlayer reaches 80 ℃, opening a valve for allowing water vapor to enter the fermentation tank, introducing the water vapor into the fermentation tank, heating the culture medium to 115 ℃, and sterilizing for 20min;
s204, stopping introducing water vapor, introducing air, cooling the tank body and the steam filter element, discharging redundant water vapor out of the fermentation tank, and automatically injecting cold water into an interlayer of the fermentation tank to gradually cool the culture medium;
s205, when the temperature is reduced to 28 ℃, opening a fermentation tank, igniting absorbent cotton with alcohol absorbed in an inoculating loop at the opening of the fermentation tank, adding 250ml of the biocontrol bacterium liquid obtained in the step 1 under the flame sterile condition, keeping the working state of an air compressor, opening a switch for intermittent stirring of the fermentation tank under the condition of ensuring continuous ventilation to the fermentation tank, and after the fermentation tank is closed for more than 24 hours, respectively obtaining bacillus subtilis after the bacterium liquid grows into a slightly turbid stateBacillus subtilis KB3 biocontrol bacterial liquid and bacillus megatheriumBacillus megaterium TZ-1 biological control bacterial liquid.
4. A method for controlling sclerotium disease of sunflower according to claim 3, wherein step S205 comprises the following steps: the intermittent stirring is specifically carried out for 2min, and the alternating circulation stirring is stopped for 5 min.
5. The method for preventing and controlling sclerotium disease of sunflower head according to claim 1, wherein the step 3 specifically comprises the steps of mixing the two biocontrol bacterial liquids obtained in the step 2 in equal proportion to obtain a composite biocontrol bacterial liquid according to 1×10 8 Concentration configuration of CFU/mL.
6. The method for preventing and controlling sclerotium disease of sunflower according to claim 1, wherein the compound biocontrol bacterial liquid obtained in the step 3 is mixed with water according to the proportion of 1:9 for dilution after 2d of the flowering period of sunflower and in the full-bloom period, and is uniformly sprayed on the surface of the flower disc of sunflower.
7. The method for preventing and controlling sclerotium disease of sunflower according to claim 6, wherein the sclerotium disease is uniformly sprayed on the surface of the flower disc of the sunflower by a spraying device, and the sunflower is planted according to wide and narrow rows: the wide row is 190m, the narrow row is 60cm, and the spraying device is used for driving between the wide rows.
8. The method for preventing and controlling sclerotium disease of sunflower head according to claim 1, wherein the step 4 specifically comprises the following steps of obtaining the composite biocontrol bacterial liquid in the step 3, and mixing the composite biocontrol bacterial liquid: the water is 1:10 are mixed and diluted in proportion and are sprayed at the inlet and outlet of beehive bees.
9. The method for preventing and controlling sclerotium disease of sunflower according to claim 8, wherein 10mL of bacterial liquid is sprayed on each beehive every 2 hours in the period of honey collection of bees in the daytime.
10. The method for preventing and controlling sclerotium disease of sunflower head according to claim 8, wherein the bacillus subtilisBacillus subtilis KB3, the strain is preserved in China general microbiological culture Collection center, and is named after classification:Bacillus subtilisthe preservation number is: CGMCC No.23108; and bacillus megateriumBacillus megaterium TZ-1, the strain is preserved in China general microbiological culture Collection center, and is named after classification:Bacillus megaterium，the preservation number is CGMCC NO.22296.
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| CN115710561A (en) * | 2022-09-03 | 2023-02-24 | 内蒙古农业大学 | Bacillus megaterium for improving alfalfa quality, screening method and application thereof |
| CN116004466A (en) * | 2023-01-07 | 2023-04-25 | 内蒙古农业大学 | Bacterial strain for preventing and treating sclerotinia rot of sunflower, screening method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115710561A (en) * | 2022-09-03 | 2023-02-24 | 内蒙古农业大学 | Bacillus megaterium for improving alfalfa quality, screening method and application thereof |
| CN115710561B (en) * | 2022-09-03 | 2024-06-14 | 内蒙古农业大学 | Bacillus megaterium for improving alfalfa quality, screening method and application thereof |
| CN116004466A (en) * | 2023-01-07 | 2023-04-25 | 内蒙古农业大学 | Bacterial strain for preventing and treating sclerotinia rot of sunflower, screening method and application |
| CN116004466B (en) * | 2023-01-07 | 2024-06-04 | 内蒙古农业大学 | Bacterial strain for preventing and treating sclerotinia rot of sunflower, screening method and application |
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