CN111197009A - Culture medium for promoting Esteya vermicola growth and spore production and preparation method thereof - Google Patents
Culture medium for promoting Esteya vermicola growth and spore production and preparation method thereof Download PDFInfo
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- CN111197009A CN111197009A CN202010028167.4A CN202010028167A CN111197009A CN 111197009 A CN111197009 A CN 111197009A CN 202010028167 A CN202010028167 A CN 202010028167A CN 111197009 A CN111197009 A CN 111197009A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention discloses a culture medium for promoting Esteya vermicola growth and sporulation and a preparation method thereof, belonging to the technical field of microbial culture. The invention discloses a culture medium for promoting Esteya vermicola growth and spore production, which comprises 230g/L of potato 170-. The culture medium disclosed by the invention shortens the growth cycle of Esteya vermicola, improves the sporulation quantity of the Esteya vermicola, and particularly improves the proportion of crescent spores with attached and lethal pine wood nematodes.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a culture medium for promoting growth and spore production of endoparasitic fungi Esteya vermicola of pine wood nematodes and a preparation method thereof.
Background
Pine nematode disease (Pine nematode) is a dangerous disease caused by Pine nematodes (Bursaphelenchus xylophilus) which rapidly wither and die and are destroyed in a piece. The disease is seriously damaged and difficult to prevent and treat, so the disease is highly valued by all countries in the world, is classified as an important dangerous forest disease, and is a key quarantine object by more than 40 national legislations. Pine wood nematodes invade the host pine body mainly through the wounds caused by adult longicorn supplying nutrition to healthy trees and laying eggs on weak trees. Pine wood nematodes originally originated in the united states, were introduced via the wood trade into japan, mainland china, korea taiwan, mexico, portugal, and spain, and developed pine wood nematode disease in succession. At present, the bursaphelenchus xylophilus disease of China spreads to 14 provinces, 192 counties (cities) and 674 towns of Jiangsu, Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Hubei, Hunan, Guangdong, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan and the like, and 500 more than ten thousand mu of pine forest is destroyed, thereby causing huge loss to national economy.
The prevention and control of the pine wilt disease become a worldwide problem, but an effective prevention and control method is lacked at present. The measures generally adopted in the world are to cut off the propagation source and the propagation path and prevent the pine wood nematode from propagating. The currently adopted method includes felling diseased wood and cutting off a propagation source; the method comprises the following steps of trapping and killing monochamus alternatus hopes by using trapping agents, spraying chemical agents such as thiacloprid, fenitrothion emulsion and the like on the air and the ground to prevent and control the monochamus alternatus hopes, applying predatory or parasitic natural enemies of the monochamus alternatus hopes such as Scleroderma spp, Dastarcus longulus, rotten wood hardwoods such as Alternaria sinensis and woodpeckers such as Dendrocops spp to prevent and control the monochamus alternatus hopes, and using fungi such as beauveria bassiana to prevent and control the monochamus alternata and the like; injecting chemical agents such as abamectin, chlorthal, aloperine as plant extract into the trunk to prevent and treat the pine wood nematode; and hybridizing the pinus massoniana, the pinus taeda and the pinus nigra to breed disease-resistant varieties. Due to the concealment of the harm of monochamus alternatus hope, chemical prevention and control are generally difficult to achieve, environmental pollution and toxic and side effects on non-target organisms can be caused, and meanwhile due to the limitations of prevention and control methods such as trunk injection and the like, a prevention and control method which is high in prevention and control efficiency, strong in operability and environment-friendly becomes an urgent need.
The microbial pesticide is used for preventing and controlling the pine wilt disease, which is a research hotspot internationally at present. Esteya vermicola is the first reported endoparasitic fungus in the pine wood nematode in the world. The strain can produce two different types of spores of crescent and rod shapes, wherein the crescent spores have high infection activity on the pine wood nematodes. Indoor plate determination, field injection test and greenhouse spraying test all verify that Esteya vermicola has excellent effect of preventing and controlling pine wood nematode.
The Esteya vermicola fungus grows slowly on the traditional solid culture, the sporulation rate is low, particularly the proportion of crescent spores which are adhered to and kill the pine wood nematodes is low, and the industrial production yield of the Esteya vermicola fungus is influenced. Therefore, it is an urgent need to solve the problems of the art to provide a culture medium for promoting the growth and spore production of Esteya vermicola, a parasitic fungus in pine wood nematodes, and a preparation method thereof.
Disclosure of Invention
In view of this, the invention provides a culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of pine wood nematodes and a preparation method thereof, which shorten the growth cycle of the Esteya vermicola, improve the sporulation rate of the Esteya vermicola and particularly improve the proportion of crescent spores with attached and lethal pine wood nematodes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of pine wood nematodes comprises the following components: 230g/L of potato 170-; the pH is 6-9.
Further, a culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of the pine wood nematode comprises the following specific components: 200g/L of potato, 20g/L of glucose, 20g/L of agar and 4-6g/L of calcium chloride; the pH is 6-9.
Further, the potato is a peeled potato.
Further, the pH was 7.
Further, a preparation method of the culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of the pine wood nematodes comprises the following specific steps:
(1) peeling and cutting potato, adding water, boiling, filtering with gauze, and collecting filtrate;
(2) adding glucose, agar, calcium chloride, and distilled water, adjusting pH, and fixing volume;
(3) the solution after constant volume is processed at 121 ℃ and 1.05X 105Autoclaving under Pa for 20min to obtain culture medium.
Further, the step (2) is added with water and boiled for 30 min.
Further, an application of the culture medium for promoting the growth and spore production of the endoparasitic fungus Esteya vermicola of the pine wood nematode in the culture of the endoparasitic fungus Esteya vermicola of the pine wood nematode is specifically as follows: inoculating Esteya vermicola to culture medium, and culturing at 23-31 deg.C for 7-14 days.
Further, the culture temperature was 26 ℃ and the culture time was 7 days.
According to the technical scheme, compared with the prior art, the invention discloses the culture medium for promoting the growth and spore production of the endoparasitic fungi Esteya vermicola of the pine wood nematodes and the preparation method thereof, the culture medium improves the growth speed of the endoparasitic fungi Esteya vermicola of the pine wood nematodes, and shortens the cultivation time; the sporulation rate of the endoparasitic fungus Esteya vermicola of the pine wood nematode is improved, particularly, the proportion of crescent spores which are adhered to and kill the pine wood nematode is obviously increased, and favorable conditions are provided for the application of the endoparasitic fungus Esteya vermicola of the pine wood nematode.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.
Example 1
A preparation method of a culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of pine wood nematodes comprises the following specific steps:
(1) peeling rhizoma Solani Tuber osi, cutting, adding 1L water, boiling for 30min, filtering with gauze, and collecting filtrate;
(2) adding 20g of glucose, 20g of agar and 5g of calcium chloride, adding distilled water, adjusting the pH value to 7, and fixing the volume to 1L;
(3) the solution after constant volume is processed at 121 ℃ and 1.05X 105Autoclaving under Pa for 20min to obtain culture medium.
Example 2
The medium was prepared according to the method described in example 1, and the pH was adjusted to 6 to 9 (Table 1).
Pouring the culture medium into a culture dish under the aseptic condition, and covering sterilized cellophane with the diameter slightly smaller than that of the culture dish after the culture medium in the culture dish is solidified; the activated endoparasitic fungus Esteya vermicola of pine wood nematode was then transferred to a medium covered with cellophane and inoculated into 10 plates. After inoculation, the cells were incubated at 26 ℃ in the dark.
After 15 days of culture, the colony diameter was measured and counted with a ruler. Fungal colonies were scraped from the solid medium covered with cellophane, and a large number of colonies were collected and divided into two portions of 5 plates each. The colonies of 5 plates were placed in an oven at 60 ℃ for two days, weighed and the fungal colony dry weight recorded. Another 5 plate colonies were transferred to a 50ml centrifuge tube, 0.5% Tween 80 was added, and several glass beads were added. Placing the mixture on an oscillator for oscillation, and counting the spore yield and the crescent spore ratio by using a blood counting plate.
TABLE 1 growth and sporulation of the fungi in the culture Medium at different pH
Note: the values in the table are mean ± sem (n ═ 3), and the upper letter after the same row data indicates significant differences at the 0.05 level.
Example 3
Referring to the preparation method of the culture medium in example 1, different metal cations (Table 2) were added to the culture medium, and the anions were chloride, KCl, CaCl2，MgCl2，FeCl2，FeCl3The same concentration of 5mg/L was set for different chlorides. 10 plates were inoculated. After the fungi are cultured for 15 days, 10 plates are divided into two parts, wherein 5 plates detect the growth speed of fungus colonies including the colony diameter and the colony dry weight, and the other 5 plates detect the sporulation quantity. Each group was subjected to 3 replicates.
TABLE 2 growth and sporulation of the fungi with different metal cations added to the culture medium
Note: the values in the table are mean ± sem (n ═ 3), and the upper letters after the data of the same row indicate significant differences at the 0.05 level; "-" indicates that colonies did not grow.
Example 4
Referring to the preparation method of the culture medium in example 1, different anions (Table 3) were added to the culture medium, the cation was calcium ion, CaCl2，CaCO3，CaSO4The same concentration of 5mg/L was set for the ionic compound. 10 plates were inoculated. After 15 days of fungus culture, 10 plates were divided into two parts, wherein 5 plates were used to detect the growth rate of fungus colonies including colony diameter and colony dry weight, and the other 5 plates were used to detect the sporulation amount and the ratio of crescent spores. Each group was subjected to 3 replicates.
TABLE 3 growth and sporulation of the fungi with different anions added to the culture
Note: the values in the table are mean ± sem (n ═ 3), and the upper letter after the same row data indicates significant differences at the 0.05 level.
Example 5
Referring to the preparation method of the medium in example 1, calcium chloride was added to the medium at various concentrations (Table 4), 0mg/L, 2mg/L, 4mg/L, 5mg/L, 6mg/L, and 8 mg/L. 10 plates were inoculated. After the fungi are cultured for 15 days, 10 plates are divided into two parts, wherein 5 plates detect the growth speed of fungus colonies including the colony diameter and the colony dry weight, and the other 5 plates detect the sporulation quantity. Each group was subjected to 3 replicates.
TABLE 4 growth and sporulation of the fungi with different concentrations of calcium chloride added to the culture medium
Note: the values in the table are mean ± sem (n ═ 3), and the upper letter after the same row data indicates significant differences at the 0.05 level.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A culture medium for promoting growth and sporulation of endoparasitic fungi Esteya vermicola of pine wood nematodes is characterized by comprising the following specific components: 230g/L of potato 170-; the pH is 6-9.
2. The culture medium for promoting the growth and sporulation of the endoparasitic fungus Esteya vermicola of the pine wood nematode according to claim 1, which is characterized by comprising the following specific components: 200g/L of potato, 20g/L of glucose, 20g/L of agar and 4-6g/L of calcium chloride; the pH is 6-9.
3. The medium for promoting the growth and sporulation of the pine wood nematode endoparasitic fungus Esteya vermicola according to claim 1 or 2, wherein the potato is a peeled potato.
4. The medium according to claim 1 or 2, wherein the pH is 7.
5. The method for preparing the culture medium for promoting the growth and sporulation of the endoparasitic fungus Esteya vermicola of the pine wood nematode according to any one of claims 1 to 4, which is characterized by comprising the following specific steps:
(1) peeling and cutting potato, adding water, boiling, filtering with gauze, and collecting filtrate;
(2) adding glucose, agar, calcium chloride, and distilled water, adjusting pH, and fixing volume;
(3) the solution after constant volume is processed at 121 ℃ and 1.05X 105Autoclaving under Pa for 20min to obtain culture medium.
6. The method for preparing a medium for promoting the growth and sporulation of Esteya vermicola, a parasitic fungus in pine wood nematodes according to claim 5, wherein the step (2) is boiling with water for 30 min.
7. The use of a medium for promoting the growth and spore production of Esteya vermicola, a parasitic fungus in pine wood nematodes, according to any one of claims 1 to 6, in the cultivation of Esteya vermicola, a parasitic fungus in pine wood nematodes by inoculating Esteya vermicola to the medium and culturing at a temperature of 23 to 31 ℃ for 7 to 14 days.
8. The use of a medium for promoting the growth and sporulation of the pine wood nematode endoparasitic fungus Esteya vermicola according to claim 7, wherein the culture temperature is 26 ℃ and the culture time is 7 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010028167.4A CN111197009A (en) | 2020-01-10 | 2020-01-10 | Culture medium for promoting Esteya vermicola growth and spore production and preparation method thereof |
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| CN202010028167.4A CN111197009A (en) | 2020-01-10 | 2020-01-10 | Culture medium for promoting Esteya vermicola growth and spore production and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114457000A (en) * | 2022-01-20 | 2022-05-10 | 中国林业科学研究院森林生态环境与自然保护研究所 | Application of wood vinegar liquid in promoting Esteya vermicola blastospore germination to generate crescent spores |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609387A (en) * | 2018-11-15 | 2019-04-12 | 华南农业大学 | A kind of fast culture process of Bursaphelenchus xylophilus inner parasitic epiphyte Esteya vermicola |
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2020
- 2020-01-10 CN CN202010028167.4A patent/CN111197009A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609387A (en) * | 2018-11-15 | 2019-04-12 | 华南农业大学 | A kind of fast culture process of Bursaphelenchus xylophilus inner parasitic epiphyte Esteya vermicola |
Non-Patent Citations (2)
| Title |
|---|
| ZHEN WANG ET AL.: "Effects of mineral salts on the growth, sporulation and virulence of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus", 《BIOCONTROL SCIENCE AND TECHNOLOGY》 * |
| 谈家金等: "几种物质对松材线虫的作用", 《四川林业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114457000A (en) * | 2022-01-20 | 2022-05-10 | 中国林业科学研究院森林生态环境与自然保护研究所 | Application of wood vinegar liquid in promoting Esteya vermicola blastospore germination to generate crescent spores |
| CN114457000B (en) * | 2022-01-20 | 2024-05-24 | 中国林业科学研究院森林生态环境与自然保护研究所 | Application of pyroligneous liquor in promoting Esteya vermicola bud conidium germination to produce crescent spore |
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