CN109497091B - Biological control preparation with nematode killing function and preparation method thereof - Google Patents
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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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/14—Boron; Compounds thereof
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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
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
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Abstract
The invention relates to the technical field of biological pesticides, in particular to a biological control preparation with a nematode killing function and a preparation method thereof. The invention provides a biological control preparation with a nematode killing function, which comprises the following main components in parts by weight: 3-7 parts of plant ash, 8-18 parts of borax and 5-10 parts of acremonium culture solution freeze-dried powder; the invention also provides a preparation method of the biocontrol preparation, which comprises the following steps: collecting culture solution after culturing Acremonium strictum, freeze-drying, and adding plant ash, borax and sawdust. The effective component in the biocontrol preparation provided by the invention is acremonium cladosporium culture solution freeze-dried powder, and the biocontrol preparation has a good effect of killing root-knot nematodes.
Description
Technical Field
The invention relates to the technical field of biological pesticides, in particular to a biological control preparation with a nematode killing function and a preparation method thereof.
Background
Root-knot nematodes (Meloidogyne) are a highly specialized species of the xenotrophic plant pathogenic nematode, male and female. The larvae are in the shape of slender worms, harm vegetables, have wide host range and can survive for one year under the condition of no host. The upper part of the damaged plant grows short and slow, the color of the leaves is abnormal, the fruit bearing is less, the yield is low, and even the plant dies in advance. The nematodes harmful to vegetables are known to be mainly high-bowknot nematodes, peanut root-knot nematodes, northern root-knot nematodes and southern root-knot nematodes. The host range of the nematode is wide, the nematode is usually harmful to more than 30 vegetables such as melons, solanaceous fruits, beans, radishes, carrots, lettuce, Chinese cabbages and the like, and the nematode can also spread some fungi and bacterial diseases. Root-knot nematodes mainly damage the roots of various vegetables, and are characterized by a more normal increase in lateral roots and fibrous roots, and the formation of spherical or conical white nodules of various sizes, sometimes in the form of marbles, on the fibrous roots of young roots. The upper part of the damaged plant grows short and slow, the color of the leaves is abnormal, the fruit bearing is less, the yield is low, and even the plant dies in advance.
At present, most methods for preventing and treating root-knot nematodes are chemical agent prevention and treatment, namely, compounds such as clomiphos, abamectin emulsifiable solution, phoxim emulsifiable solution, thiamethoxam water dispersible granules and the like are applied to crop soil, but chemical prevention and treatment often causes irreparable damage to the soil environment, and chemical insecticidal methods cause environmental pollution and harm to human health. The main management measures for preventing and controlling the root-knot nematodes are field rotation and use of non-host plants or disease-resistant plants, but the method has certain limitations, for example, most species of the root-knot nematodes have wider host range, and meanwhile, most species of the root-knot nematodes which are pathogenic in the field are mixed populations of several species, so that the prevention and control effect is difficult to maintain. Other measures such as sun exposure and flooding, using disease-free nursery stocks and soil improvement can achieve some control effects under certain conditions, but the effects are little or have great limitations. Therefore, the pollution-free sustainable treatment method taking biological control as a core is a main direction for controlling the root-knot nematode disease.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a biological control preparation with a nematicidal function and a preparation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a biological control preparation with a nematode killing function comprises the following components in parts by weight: 3-7 parts of plant ash, 8-18 parts of borax and 5-10 parts of Acremonium culture solution freeze-dried powder, wherein the Acremonium is classified as Acremonium yunnanensis and is preserved in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms in 2016, 4-21 months, the preservation number is CGMCC No.12374, and the preservation unit address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
Further, the biocontrol agent with the nematicidal function is powder.
The biological control preparation with the nematode killing function can be applied to killing root-knot nematodes of crops.
Further, the application method of the biological control preparation with the nematicidal function in killing the root-knot nematodes of the crops comprises the following steps: 2L of water is added into every 50g of the biocontrol agent, and the biocontrol agent is uniformly mixed and irrigated into the soil at the roots of the crops.
The invention also provides a preparation method of the biological control preparation with the function of killing nematodes, which comprises the following steps:
culturing acremonium
Inoculating acremonium cladosporioides on a PDA (personal digital assistant) plate culture medium, culturing for 5-7 days at the temperature of 28 ℃, inoculating acremonium cladosporioides cultured on the PDA plate culture medium into 100mL of LB liquid culture medium in an ultra-clean workbench at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way, and inoculating 100mL of a bacterial liquid obtained by culture into 1L of sterile LB liquid culture medium at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way;
② obtaining culture solution freeze-dried powder
Centrifuging the Acremonium strictum bacterial liquid obtained in the step I, rotating at 12000rpm for 10min, reserving supernate after centrifugation, removing precipitates, filtering the supernate by using a 0.45-micron filter, and freeze-drying at low temperature until the water content is lower than 13%;
③ obtaining the biocontrol agent
And (4) taking 5-10 parts of the culture solution freeze-dried powder of acremonium strictum obtained in the step (II), 3-7 parts of plant ash and 8-18 parts of borax to obtain the biocontrol preparation with the nematode killing function.
Has the advantages that:
(1) the biocontrol preparation provided by the invention takes culture solution freeze-dried powder for culturing acremonium ramosum as a main active ingredient, does not contain active bacteria, has little influence on the original soil flora of crops, and is suitable for cultivating crops which need not to be changed but need to kill root-knot nematodes and have proper soil environment flora;
(2) the biological control preparation is added with plant ash and borax, which is beneficial to the active substances in the biological control preparation to play a role and balance the pH value of soil;
(3) experiments prove that the killing rate of the acremonium culture solution to the root-knot nematode is 100% after the acremonium culture solution is diluted by 20 times, and the death rate of the root-knot nematode is reduced along with the increase of the dilution times, which indicates that the acremonium culture solution secretes active substances capable of killing the root-knot nematode in the growth and proliferation process; after the tomato root-knot nematodes are cultured in the Acremonium culture solution for 48 hours, the nematodes become straight, cavities are formed in the nematodes, and the nematodes begin to die, and the culture solution has the effect of killing the root-knot nematodes;
(4) the biocontrol agent disclosed by the invention is not added with chemical components, has small environmental pollution and accords with the modern agricultural concept of sustainable development.
Drawings
FIG. 1 shows graphs of Acremonium chrysosporium culture solution for killing nematodes
In the figure, A is a picture of normal tomato root-knot nematode; and B is a picture of the tomato root-knot nematode cultured by the Acremonium strictum culture solution for 48 hours, wherein the arrow points to a cavity formed in the tomato root-knot nematode.
FIG. 2 is a statistical plot of nematode mortality.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows: preparation of biological control preparation with nematode killing function
Culturing acremonium
Inoculating acremonium cladosporioides on a PDA (personal digital assistant) plate culture medium, culturing for 5-7 days at the temperature of 28 ℃, inoculating acremonium cladosporioides cultured on the PDA plate culture medium into 100mL of LB liquid culture medium in an ultra-clean workbench at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way, and inoculating 100mL of a bacterial liquid obtained by culture into 1L of sterile LB liquid culture medium at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way;
② obtaining culture solution freeze-dried powder
Centrifuging the Acremonium strictum bacterial liquid obtained in the step I, rotating at 12000rpm for 10min, reserving supernate after centrifugation, removing precipitates, filtering the supernate by using a 0.45-micron filter, and freeze-drying at low temperature until the water content is lower than 13%;
③ obtaining the biocontrol agent
And (4) taking 5-10 parts of the culture solution freeze-dried powder of acremonium strictum obtained in the step (II), 3-7 parts of plant ash and 8-18 parts of borax to obtain the biocontrol preparation with the nematode killing function.
Example one of the biological control agents is a powder which is mixed with water and irrigated to the soil at the root of the crop to achieve the effect of killing nematodes, and the obtained biological control agent is preferably stored in an environment below 4 ℃.
Example two: study on insecticidal action of culture solution of acremonium subtense
1 isolated culture of nematodes
a. Cleaning tomato roots containing root-knot nematodes, cutting into pieces with scissors, and pulverizing into pieces.
b. Pouring the crushed root material into a nematode screening sample sieve (the upper part is 320 meshes, the lower part is 500 meshes, and the two are overlapped together) to screen nematode eggs, washing the crushed root material in a proper amount of running water (the sieve is obliquely flushed with water) for 5 minutes, enabling nematode egg blocks to leak into a second sieve, washing the nematode eggs in the 500 meshes sieve into a culture dish by using a washing bottle, and repeatedly screening once.
c. Observing whether nematode eggs and the number of the eggs are screened under a stereoscope, if the nematode eggs are cultured, putting the nematode eggs into a incubator at the temperature of 28 ℃ for culture, periodically adding water, incubating the nematode for 4-7 days, observing the incubation condition of the nematode eggs, wherein most of the nematode is incubated, and if the number of the nematode eggs is sufficient, carrying out the next experiment.
2 obtaining Acremonium culture solution
a. Acremonium culture
Acremonium terricola cultured on PDA plate medium (cultured for 5-7 days) was inoculated into 100mL of LB liquid medium in a clean bench and cultured in a shaker at 180rpm and 28 ℃ for three days. Inoculating the cultured bacterial liquid into 1L of sterile LB liquid medium, shaking at 180rpm and 28 deg.C, and performing amplification culture for three days, and observing the growth condition of the bacteria after three days.
b. Extraction of Acremonium culture solution
And (3) putting the acremonium strictum cultured for three days into a large centrifuge (12000rpm, 10min) for centrifugation, discarding the precipitate after centrifugation, reserving the supernatant, sucking the supernatant by using a 5mL syringe, filtering by using a large filter head, collecting the filtered acremonium strictum extracting solution, and storing in a refrigerator at 4 ℃ for later use.
3-branch acremonium culture solution for culturing nematodes
Culturing the nematodes by using the acremonium strictum culture solution obtained in the step 2, and observing the growth condition of the nematodes
a. Diluting the acremonium culture solution by the following dilution times: 10. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times 19 concentrations. The diluted bacteria liquid is put into a 24-well plate according to the concentration sequence, 1mL of diluted culture solution is put into each well, and 1mL of sterile water is added into the last group as a control.
b. And (3) sucking the nematodes in the culture dish by using a 10uL liquid-transfering gun to put diluted bacteria liquid for culture, ensuring that the number of the nematodes sucked each time is approximately equal, and adding about 50 nematodes into every 1mL of solution. After the solution nematodes with different concentrations are put in the culture box, the solution nematodes are placed under a stereoscope to detect whether the number of the nematodes is proper or not, if the number is proper, the solution nematodes are placed in an incubator at 28 ℃ for culture, and the nematode growth condition is regularly observed.
c. And (3) regularly observing the growth condition of the nematodes in the acremonium cladosporioides extracting solution, and counting the mortality of the nematodes after 48 hours.
3 results
As shown in fig. 1, a diagram A is the nematode morphology of the acremonium obliquum culture solution in a normal state, a diagram B is the nematode morphology after 48 hours of culture in the acremonium obliquum culture solution, arrows point to cavities appearing in the nematodes, the body of the nematodes becomes straight after the nematodes are cultured, the cavities are formed in the nematodes, and the nematodes begin to die; as shown in FIG. 2, the mortality rate of nematodes was close to 100% at 10 and 20 times of dilution, and decreased with increasing dilution, but the culture broth dilution still had significant nematicidal effect compared to the control.
In the second example, experiments of culturing nematodes with an acremonium culture solution prove that the acremonium culture solution contains active substances secreted by acremonium and having a nematode killing function, and a biocontrol agent added with freeze-dried powder of the acremonium culture solution has a nematode killing effect.
The present invention is not described in detail, but is known to those skilled in the art. Finally, the above embodiments are merely illustrative and not restrictive, and modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is to be covered by the claims.
Claims (5)
1. A biological control preparation with nematicidal function is characterized in that: comprises the following components in parts by weight: 3-7 parts of plant ash, 8-18 parts of borax and 5-10 parts of acremonium strain culture solution freeze-dried powder;
the Acremonium yunnanensis is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms at 2016, 4, 21 days, with the preservation number of CGMCC No.12374 and the preservation unit address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
2. The nematicidal biocontrol agent of claim 1, wherein: the biocontrol agent is powder.
3. The use of the nematicidal biocontrol agent of claim 1 to kill root-knot nematodes in crops.
4. The use of a nematicidal biocontrol agent to kill root-knot nematodes in crops according to claim 3, wherein: the application method comprises the following steps: 2L of water is added into every 50g of the biocontrol agent, and the biocontrol agent is uniformly mixed and irrigated into the soil at the roots of the crops.
5. The method for preparing a biological control agent with nematicidal effect of claim 1, comprising the steps of: the method comprises the following steps:
culturing acremonium
Inoculating acremonium cladosporioides on a PDA (personal digital assistant) plate culture medium, culturing for 5-7 days at the temperature of 28 ℃, inoculating acremonium cladosporioides cultured on the PDA plate culture medium into 100mL of LB liquid culture medium in an ultra-clean workbench at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way, and inoculating 100mL of a bacterial liquid obtained by culture into 1L of sterile LB liquid culture medium at the rotating speed of 180rpm and the temperature of 28 ℃ for three days in a shaking way;
② obtaining culture solution freeze-dried powder
Centrifuging the Acremonium strictum bacterial liquid obtained in the step I, rotating at 12000rpm for 10min, reserving supernate after centrifugation, removing precipitates, filtering the supernate by using a 0.45-micron filter, and freeze-drying at low temperature until the water content is lower than 13%;
③ obtaining the biocontrol agent
And (4) uniformly mixing 5-10 parts of the culture solution freeze-dried powder of acremonium strictum obtained in the step (II), 3-7 parts of plant ash and 8-18 parts of borax to obtain the biocontrol preparation with the nematode killing function.
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|---|---|---|---|---|
| CN101235355A (en) * | 2008-03-03 | 2008-08-06 | 中国热带农业科学院环境与植物保护研究所 | Plants endogenetic fungus and application thereof |
| CN103667083A (en) * | 2013-12-19 | 2014-03-26 | 陕西省微生物研究所 | Acremonium, culturing method of acremonium and application of acremonium to preparation of original spore powder of meloidogyne pesticide |
| CN104593266A (en) * | 2013-11-04 | 2015-05-06 | 中国农业科学院蔬菜花卉研究所 | Tomato endophytic fungi acremonium implicatum and applications thereof in biocontrol of tomato root knot nematode disease |
| CN105907648A (en) * | 2016-05-16 | 2016-08-31 | 杜云龙 | Preparation method and application of acremonium strictum of endophytic fungi of panax notoginseng |
| CN105993871A (en) * | 2016-05-16 | 2016-10-12 | 杜云龙 | Rapid seedling-raising method capable of promoting high germination rate of pseudo-ginseng by utilizing acremonium strictum |
-
2018
- 2018-12-27 CN CN201811611290.8A patent/CN109497091B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235355A (en) * | 2008-03-03 | 2008-08-06 | 中国热带农业科学院环境与植物保护研究所 | Plants endogenetic fungus and application thereof |
| CN104593266A (en) * | 2013-11-04 | 2015-05-06 | 中国农业科学院蔬菜花卉研究所 | Tomato endophytic fungi acremonium implicatum and applications thereof in biocontrol of tomato root knot nematode disease |
| CN103667083A (en) * | 2013-12-19 | 2014-03-26 | 陕西省微生物研究所 | Acremonium, culturing method of acremonium and application of acremonium to preparation of original spore powder of meloidogyne pesticide |
| CN105907648A (en) * | 2016-05-16 | 2016-08-31 | 杜云龙 | Preparation method and application of acremonium strictum of endophytic fungi of panax notoginseng |
| CN105993871A (en) * | 2016-05-16 | 2016-10-12 | 杜云龙 | Rapid seedling-raising method capable of promoting high germination rate of pseudo-ginseng by utilizing acremonium strictum |
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