CN112868671A - Preparation method and application of killed bacillus vallismortis suspending agent for killing citrus mites - Google Patents
Preparation method and application of killed bacillus vallismortis suspending agent for killing citrus mites Download PDFInfo
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Images
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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
- 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
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
- A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
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- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
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Abstract
The invention belongs to the technical field of pesticides, and particularly relates to a preparation method and application of a killed bacillus vallismortis suspending agent for killing citrus mites. The suspending agent is Bacillus cereus (Bacillus cereus) to be killedBacillus vallismortis) Mixing the NBIF-001 fermentation liquid with sodium lignosulfonate, xanthan gum, potassium sorbate and glycol. The suspending agent prepared by the invention has good mite killing effect on citrus red mites and rust ticks, is a high-efficiency biological pesticide, and has good application and popularization prospects.
Description
Technical Field
The invention belongs to the technical field of pesticides, and particularly relates to a preparation method and application of a killed bacillus vallismortis suspending agent for killing citrus mites.
Background
The citrus mites belong to the Araneid, and mainly comprise Tetranychus, gall mite and Tarsonemus tarsalon. The tetranychus urticae (Panonychus citri), Henychus citri (Eotetranychus kankitus) and Henychidae (Schizotarychus palmatrae) are common spider mites in China. The gall mite is the tick orange rust (Eriophyes oleivorus). Tarsonemus dorsalis (Polyhagotarsonemus latus) is present on the lateral side of Tarsonemus tarsonemus. The harmful mites are all sucked by a piercing-sucking mouthpart. The harm to hosts is wide, such as citrus, loquat, grape, cherry, peach, pear, strawberry and the like, and the harm to citrus is serious. Tetranychus urticae is mainly harmful to leaves, twigs, young fruits, flower buds and other parts of hosts, and is seriously damaged by tender leaves and tender tips. The damaged parts are faded green firstly and then appear grey-white spots, leaves lose luster, damaged fruits are grey-white, a large amount of fallen leaves, fallen flowers and fallen fruits are caused in serious conditions, and the yield and the quality of the oranges are greatly influenced. After the gall mites are damaged, yellow green plaques and scabs appear on the damaged parts of young leaves firstly, the damaged parts are deformed to form hair galls, host tissues in the hair galls generate grey-white villi due to stimulation, and the villi gradually become yellow brown, reddish brown to dark brown and are shaped like felt. Tarsonemus tarsaloides mainly damages tender shoots and young fruits of oranges, and young adult mites gather on the back surfaces of the shoots and leaves and the surfaces of the young fruits to suck juice, damage epidermal tissues, enable damaged tender leaves to curl back, harden and become small leaf stalks, lose the young shoots and the sprouting capacity, and become bald and growth-hindered. After the young fruit is damaged, the peel loses the luster and forms brown spots because the epidermis and oil vacuole are damaged, and when the young fruit is seriously damaged, the whole fruit is damaged to become small fruit and lose the commodity value.
The method for preventing and controlling citrus mites mainly comprises agricultural prevention and control, biological prevention and control and chemical prevention and control. The agricultural control comprises cleaning the garden in winter, cleaning the diseased leaves and burning the diseased leaves intensively, and reducing the overwintering number of the pest mites; strengthen the management of fertilizer and water, improve the microclimate of the orchard, and the like. The biological control comprises the control by utilizing green and safe means such as natural enemies, biological acaricides and the like. Chemical control is mainly carried out by using chemical acaricide and other medicaments. In recent years, because the variety of acaricides is lack of substitution, the acaricides are repeatedly used in large quantity, the generation of resistance to harmful mites is accelerated, the mite damage is more and more serious, and great economic loss is caused to growers.
Killed Bacillus vallismortis (Bacillus vallismortis) is an aerobic, cell-producing gram-positive bacterium. It was first isolated from soil in the united states dead valley national park by american biologist Michael s. roberts in 1996 and was named dead agrobacillus. The bacillus subtilis is similar to bacillus subtilis, but the fatty acid composition and the DNA sequence of the bacillus subtilis are different from those of the bacillus subtilis. At present, few documents are internationally reported in the research on killed bacillus vallismortis, the research is mainly focused on the aspects of affinity analysis, bacteriostatic activity, degradation activity, bioadsorption activity, enzyme production and the like, the environmental stress resistance is strong, the biological activity is excellent, and the method can be widely applied to the fields of agriculture, medicine, environmental management and the like.
The killed bacillus cereus NBIF-001 is a proprietary strain disclosed by the applicant, and can be used for preventing and treating watermelon fusarium wilt, cucumber gray mold, rice sheath blight and mite inhibition; meanwhile, the strain can also inhibit fungi including tomato fusarium wilt, tomato early blight, fusarium graminearum, wheat fusarium graminearum or rice sheath blight.
The invention mainly aims at the NBIF-001 strain to create and apply different dosage forms, a new product is put on the market, and the difference of the dosage forms has a decisive effect on the performance of the product. The insecticidal effect of products with different dosage forms is different for the same strain, for example, the research and application of dosage forms of mosquito-killing bacillus pumilus (Chen national English, 2006), the experimental result shows that the titer of the floating granule Bti is 550IU/mg, the titer of the floating block Bti is 230IU/mg, and the titer of the foaming agent Bti is 513IU/mg, thus proving the influence of different dosage forms on the drug effect.
The specific formulation of the formulation also has an important influence on the biocontrol effect of the strains, such as different stabilizers, dispersants or antifreeze agents, and has a significant influence on the biocontrol effect of different strains, that is, the formulation of the auxiliary materials has an influence on the main active ingredients. For example, the Chinese invention patents 'tetranychus gossypii and bacillus thuringiensis killing suspending agent, preparation method and application' and 'grub pest killing bacillus thuringiensis microcapsule suspending agent and preparation method' relate to microbial suspending agent products with different formulas, and have obvious influence on drug effect.
The same medicament has different control effects on different pest mites, for example, the control effect on citrus rust tick is 99.79 percent 28 days after 240g/L spirodiclofen emulsifiable concentrate is taken, and the control effect on citrus red spiders is 79.42 percent (Liushawu, etc., 2018); the death rate of citrus red mites 7 days after the 15 percent pyridaben emulsifiable solution is 19.68 percent, and the death rate of citrus rust tick is 56.79 percent. Therefore, how to make a product into the most effective dosage form is crucial.
At present, no related formulation product developed aiming at the dead bacillus vallismortis exists internationally, so that on the basis of the background, the dead bacillus vallismortis bacillus suspending agent is required to be developed, is used in the field of citrus mite damage prevention and control, and has important production practice significance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method and application of a bacillus vallismortis suspending agent for killing citrus harmful mites, which is used for preventing and controlling citrus red spiders and ticks so as to achieve the ecological benefits of more effectively preventing and controlling the harmful mites and being more friendly to human bodies and environmental organisms.
In order to solve the technical problems, the invention adopts the following technical scheme:
the killed bacillus vallismortis suspending agent for killing citrus mites comprises the following components in parts by weight: 70-90 parts of dead bacillus vallismortis fermentation stock solution, 5-10 parts of sodium lignosulphonate, 0.5-1.5 parts of xanthan gum, 2-4 parts of potassium sorbate and 2-5 parts of ethylene glycol;
after the preparation is finished, the effective bacterium concentration of dead bacillus vallismortis in the suspending agent is 80-150 multiplied by 108CFU/mL；
The dead Bacillus vallismortis NBIF-001 with the preservation number of CCTCC NO: m2015087.
The fermentation stock solution of the dead bacillus vallismortis is prepared by fermenting conventional dead bacillus vallismortis at present.
The preparation method of the bacillus vallismortis suspending agent for killing citrus mites comprises the steps of adding the components into bacillus vallismortis fermentation stock solution, and uniformly stirring.
The application of the bacillus vallismortis suspending agent for killing citrus harmful mites in preventing and controlling the citrus harmful mites, particularly red spiders, ticks, tetranychus cinnabarinus or tetranychus urticae.
Compared with the prior art, the invention has the following technical effects:
the suspending agent is the first creation of a killed bacillus vallismortis dosage form, has more thorough effect of preventing and treating target mites and is safer for users and environmental organisms compared with the conventional medicament. The invention can achieve the ecological benefit of more effectively preventing and controlling the pest mites and being more friendly to human bodies and environmental organisms. The field control effect test result shows that the acaricide has excellent acaricidal activity on citrus red spiders and rust ticks, and can be popularized and used as an effective medicament for field control.
The invention provides a special adaptation for dead Bacillus vallismortis NBIF-001 and a suspending agent formula thereof for bio-control of citrus red spiders and tick mites.
Drawings
FIG. 1 is a graph of the control effect of citrus rust ticks;
wherein, A, citrus before administration is not used; B. orange 7 days after administration; C. citrus is administered 14 days after administration.
Detailed Description
The technical scheme of the invention is a conventional operation method in the field if not specifically stated, and the reagent or the material is sourced from commercial sources if not specifically stated.
The dead Bacillus vallismortis NBIF-001 in the embodiment of the invention, and the preservation number is CCTCC NO: m2015087, disclosed in patent application No. 2015104854956.
The preparation method of the dead bacillus vallismortis fermentation stock solution comprises the following steps:
the fermentation medium is prepared from (by weight) corn starch 2%, soybean meal 4%, yeast powder 1.5%, fish meal 1.5%, pH7.0, and water in balance.
The fermentation was carried out in a 200L fermenter (120L pot volume) with a ratio of aeration per minute to fermenter volume of 1.5: 1, the culture temperature is 32 ℃, the inoculum size is 3 percent, the rotating speed is 150r/min, and the fermentation time is 38 h.
After the fermentation is finished, the effective bacteria concentration in the fermentation stock solution is 150 multiplied by 108CFU/mL, used in the following examples.
Example 1:
preparation of citrus pest mite killing bacillus cereus suspension A (in the embodiment of the invention or simply referred to as 001 suspension A):
84g of dead bacillus vallismortis fermentation stock solution, 10g of sodium lignosulphonate, 1.5g of sodium carboxymethyl cellulose, 2.5g of potassium sorbate and 2g of ethylene glycol.
The raw materials are premixed according to the formula proportion and fully mixed and stirred.
Example 2:
preparing a bacillus vallismortis suspending agent B for killing citrus mites and killing death germs:
001 suspending agent B1:
84g of dead bacillus vallismortis fermentation stock solution, 10g of sodium lignosulphonate, 1.5g of xanthan gum, 2.5g of potassium sorbate and 2g of ethylene glycol;
001 suspending agent B2:
70g of dead bacillus vallismortis fermentation stock solution, 5g of sodium lignosulphonate, 0.5g of xanthan gum, 2g of potassium sorbate and 2g of ethylene glycol;
001 suspending agent B3: 90g of dead bacillus vallismortis fermentation stock solution, 10g of sodium lignosulphonate, 1.5g of xanthan gum, 4g of potassium sorbate and 5g of ethylene glycol;
the raw materials are premixed according to the formula proportion and fully mixed and stirred.
Example 3:
preparation of citrus pest mite killing bacillus cereus suspension C (in the embodiment of the invention or simply referred to as 001 suspension C):
84g of dead bacillus vallismortis fermentation stock solution, 10g of sodium lignosulphonate, 1.5g of xanthan gum, 2.5g of sodium benzoate and 2g of ethylene glycol.
The raw materials are premixed according to the formula proportion and fully mixed and stirred.
Example 4:
indoor activity determination of the citrus pest mite killing killed bacillus vallismortis suspending agent on tetranychus cinnabarinus and tetranychus urticae:
the test agents are 3% of abamectin emulsifiable concentrate (Hebei Bayuan biological products, Inc.), 15% of pyridaben emulsifiable concentrate (Shandong Henlida biological technology, Inc.), 40% of propargite emulsifiable concentrate (Qingdao Han Biotechnology, Inc.), 200g/L of chlorantraniliprole suspending agent (Meiguo Mei Co., Ltd.) and killed bacillus vallismortis wettable powder A, B, C. The pathogen is Tetranychus cinnabarinus and Tetranychus urticae.
The method comprises the following operation steps: reference is made to the standard method recommended by FAO (food and agriculture organization of the united nations) for the determination of mites-the glass slide immersion method. The double-sided adhesive tape is cut into 2-3cm long, the double-sided adhesive tape is attached to one end of a microscope slide, paper pieces on the adhesive tape are removed by tweezers, female adult mites which are consistent in size, bright in body color and lively in action are selected by a zero-number brush pen, the backs of the female adult mites are adhered to the double-sided adhesive tape (paying attention to the fact that the female adult mites do not stick to the mite feet, the mite beards and a mouth tool), 4 lines of the double-sided adhesive tape are adhered to each piece, and 10 heads of. Placing in a biochemical incubator with the temperature of 25 ℃ and the relative humidity of about 85% for 4h, observing by using binoculars, and removing dead or inactive individuals. Diluting the medicament with water for 5-7 concentrations on the basis of pre-test, soaking one end of the glass slide with the mites into the medicinal liquid, slightly shaking for 5s, taking out, and quickly sucking away the mites and the redundant medicinal liquid around the mites with absorbent paper. Placing in the biochemical incubator, and inspecting the result by using binoculars after 24 h. The mites were touched with a brush pen and died if they were immobile. Each concentration was repeated 3 times, and the control was made by soaking in clean water.
The results of the bioassay of Tetranychus cinnabarinus by each agent according to the above experimental procedures are shown in Table 1 below. LC (liquid Crystal)50Calculation of value by SPASS 19.0 data processing software.
Among them, the results in tables 1 and 2 are the results of the mite killing measurement performed immediately after the fermentation liquids were obtained and each agent was prepared. Table 3 shows changes in acaricidal activity of the prepared agents after 6 months of storage.
TABLE 1 miticidal Activity of each agent against Tetranychus cinnabarinus
TABLE 2 acaricidal Activity of the Agents against Tetranychus urticae
TABLE 3 acaricidal Activity of each agent against Tetranychus urticae after 6 months of storage at room temperature
Example 5:
the field activity of the citrus pest mite killing bacillus vallismortis suspending agent on citrus red spiders is determined as follows:
the test is carried out in a citrus orchard in Ganzhou Dayun county in Jiangxi province in 6 months in 2020, the soil type of the region belongs to clay, the soil fertility is moderate, and the pH value is 6.1. The tree ages 2 years, the plant spacing is 1.5m, the row spacing is 2.5m, the tree height is 1.0-1.3 m, and the crown diameter is 0.5-0.8 m. The average temperature is 10-21 ℃ and the atmospheric humidity is 25%. The cultivation period, the growth period and the density of each treatment are consistent, intertillage weeding, water fertilizer and the like are managed according to the conventional method, and no other medicament is used in the test period. The test is carried out in 23 days after 6 months, the field test insects are in the advanced adult mite stage during the test, the leaves are investigated by using a hand-held magnifier during the test, and the occurrence conditions of red spiders in the test area are basically consistent.
Reagent to be tested: 20% of abamectin spirodiclofen suspending agent (Hebei Xinbai agricultural science and technology Limited, 3000 times diluted); the biological acaricide NBIF-001 suspension A, B1, wherein the effective bacterial concentration of A and B1 is 100 hundred million CFU/mL, and is diluted 100 times with water when in use.
The test was performed for a total of 4 treatments, the doses of which are shown in table 1, each treatment was repeated 3 times, randomized in blocks, 3 plants were administered per cell, and protective rows were placed around. Spraying the medicine in 23 days (sunny day) in 6 months at night, spraying the tree crown with a controllable pressure manual sprayer, and uniformly spraying the front and back surfaces of the leaves to make them fully contact with the medicine liquid, preferably without dripping the medicine liquid. Three citrus trees are taken in each cell, lateral branches are marked in east, west, south, north and middle 5 directions of the trees, the number of red spiders on the front and back sides of 25 leaves is investigated totally, and all live mites are directly observed and recorded by a hand-held magnifier. The base number of the study was determined before administration, and the study was performed 2 times after administration for 1 day and 4 days.
The drug effect calculation method comprises the following steps: the prevention effect is calculated according to the base number of red spiders before the medicine and the number of the red spiders surviving each day after the medicine, and the calculation formula is shown as follows. The data were analyzed for significance using the DPS software Duncan's new repolarization differences.
The results of the field control effect of several miticides on citrus red mites are shown in table 3.
TABLE 4 efficacy results of several acaricides for controlling citrus red spider mites
Example 6:
the field activity of the citrus pest mite killing bacillus vallismortis suspending agent on citrus rust ticks is determined as follows:
the test is carried out in a citrus orchard in a golden water region of Wuhan city, Hubei province in 2019 and 10 months, the soil type of the region belongs to yellow brown soil, and the soil fertility is moderate. The tree is 5 years old, the plant spacing is 1.0m, the row spacing is 2.0m, the tree height is 2.0-3.0 m, and the crown diameter is 1.0-2.0 m. The average temperature is 15-21 ℃ and the atmospheric humidity is 45%. The cultivation period, the growth period and the density of each treatment are consistent, intertillage weeding, water fertilizer and the like are managed according to the conventional method, and no other medicament is used in the test period. The pesticide is applied 10 days after 10 months, the field test insects are in the advanced adult mite stage during the test, the leaves are investigated by using a hand-held magnifier during the test, and the occurrence conditions of the rust ticks in the test area are basically consistent.
Reagent to be tested: 15% pyridaben emulsifiable concentrate (Shandong Henglida Biotech Co., Ltd., 2500-fold dilution); biological acaricide NBIF-001100 hundred million CFU/mL suspension B1 (75-fold dilution with water at the time of use).
The test is carried out for 3 times, each treatment is repeated for 3 times, the treatment is randomly arranged in blocks, 10 plants are applied to each cell, and protective rows are arranged around the cells. Spraying the medicine in 10 months and 10 days (sunny day) evening, spraying the tree crown with a controllable pressure manual sprayer, and uniformly spraying the front and back surfaces of the leaves to make the leaves fully contact with the medicine liquid, wherein the medicine liquid is preferably not dropped. Healthy and diseased fruit counts per tree were investigated before and 12d after drug administration, respectively.
The drug effect calculation method comprises the following steps: the relative prevention effect is calculated according to the disease and fruit rate before and after the medicine. The calculation formula is as follows. The data were analyzed for significance using the DPS software Duncan's new repolarization differences.
The results of the field control of several acaricides against citrus rust ticks are shown in table 4.
TABLE 4 efficacy results of several acaricides against Citrus reticulata rust tick
Example 7:
citrus fruit state observation
The change of the citrus fruit disease after the drug administration (shown in figure 1) is observed, and the result shows that: after the 001 suspending agent is used, the browning speed of fruits is greatly reduced, the quantity of the fruits is reduced, and a certain greening effect is achieved on leaves.
Claims (3)
1. The killed bacillus vallismortis suspending agent for killing citrus mites comprises the following components in parts by weight: 70-90 parts of dead bacillus vallismortis fermentation stock solution, 5-10 parts of sodium lignosulphonate, 0.5-1.5 parts of xanthan gum, 2-4 parts of potassium sorbate and 2-5 parts of ethylene glycol;
the effective bacterium concentration of killed glutamic bacillus in the suspending agent is 80-150 multiplied by 108 CFU/mL；
The dead Bacillus vallismortis NBIF-001 with the preservation number of CCTCC NO: m2015087.
2. Use of the suspension of claim 1 in the preparation of a pesticide for citrus mites.
3. The use according to claim 2, wherein the citrus pest mites are red spider mites, ticks, tetranychus cinnabarinus or tetranychus urticae.
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