• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present invention discloses a culture medium for increasing a growth rate and stress 5 resistance of Esteya vermico/a and a preparation method of the culture medium and belongs to the technical field of microbial culture. The culture medium for increasing the growth rate and stress resistance of Esteya vermico/a disclosed in the present invention includes 170 - 230 g/L of potatoes, 15 - 25 g/L of glucose, 17 - 23 g/L of agar, 0.05 - 0.15 g/L of glycine and 4 - 6 g/L of calcium chloride. The culture medium in the present invention can shorten a growth cycle of 10 Esteya vermico/a, increase spore production of the Esteya vermico/a, particularly increase a ratio of crescent spores capable of adhering and causing death of Bursaphe/enchus xylophi/us, and increase drought stress and ultraviolet stress resistance of the spores in a natural environment.
    公开号:NL2025554A
    申请号:NL2025554
    申请日:2020-05-11
    公开日:2021-08-30
    发明作者:Zhang Xiangyao；Zhang Bo；Zhao Zhilong；Li Xinpeng；Shi Xiaowei；Xin Jie；Wang Zhen；Kuang Pengqun；Li Yongxia
    申请人:Univ Linyi；
    IPC主号:
    专利说明:

    CULTURE MEDIUM FOR INCREASING GROWTH RATE AND STRESS RESISTANCE OF
    ESTEYA VERMICOLA AND PREPARATION METHOD OF CULTURE MEDIUM Technical Field The present invention relates to the technical field of microbial culture, and particularly relates to a culture medium for increasing a growth rate and stress resistance of a parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus and a preparation method of the culture medium. Background Pine wilt disease is a dangerous disease capable of causing rapid withered death and continuous destruction of pine trees caused by Bursaphelenchus xylophilus. The pine wilt disease is highly valued by various countries in the world due to severe hazards and control difficulty, is listed as an important hazardous forest disease, and is legalized as a key quarantine object by more than 40 countries. The Bursaphelenchus xylophilus mainly invades host pine trees via wounds caused by adult longhorn beetles that supplement nutrition on healthy trees and lay eggs on aged trees. At present, the pine wilt disease in China has spread to 14 provinces such as Jiangsu, Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Hubei, Hunan, Guangdong, Guangxi, Chongqing, Sichuan, Guizhou and Yunnan, 192 counties (cities) and 674 villages and towns, destructs pinewoods of more than 5 million mu, and causes heavy losses to national economy. Control of the pine wilt disease has become a worldwide problem, but an effective control method does not exist at present. General measures taken in the world are to cut off propagation sources and propagation paths and stop propagation of Bursaphelenchus xylophilus. Methods that are currently adopted include cutting down diseased woods and cutting off propagation sources; trapping and killing Monochamus alternatus by utilizing trappers, spraying chemical agents such as thiacloprid and fenitrothion emulsion in the air and on the ground to control long - horned beetles, controlling the long - horned beetles by using predatory or parasitic natural enemies of the long - horned beetles such as Scleroderma spp., Dastarcus longulus, Allecula fuliginosa and Dendrocops spp. , and controlling the long - horned beetles by utilizing beauveria bassiana and other fungi; injecting chemical agents to tree trunks such as abamectin, Chongxianging and plant extract aloperin to control Bursaphelenchus xylophilus; breeding disease - resistant varieties by hybridizing masson pine, loblolly pine and Japanese black pine, and the like. Due to concealment of hazards of the Monochamus alternatus, chemical control generally difficultly comes into effect, and environmental pollution and toxic or side effects to non - target organisms are caused. Meanwhile, due to limitations of the control methods such as tree trunk injection, a control method that is high in control efficiency, high in operability, green and environment - friendly becomes an urgent need. Control of the pine wilt disease by utilizing microbial pesticides is a current international research hotspot. Esteya vermicola is a parasitical fungus in Bursaphelenchus xylophilus reported first in the world. The fungus may produce spores of two different types, that is, crescent and rodlike, wherein the crescent spores have infection activity on the Bursaphelenchus xylophilus. Indoor plate determination, field injection test and greenhouse spray test prove that the Esteya vermicola has an excellent effect of controlling the pine wilt disease.
    The fungus Esteya vermicola is low in growth rate in traditional solid culture and low in spore production rate. Particularly, a ratio of the crescent spores capable of adhering and causing death of Bursaphelenchus xylophilus is low, and an industrial production rate of the fungus Esteya vermicola is influenced. Meanwhile, the spores are affected by drought, high temperature, ultraviolet irradiation and the like in the natural environment, so that a germination rate and a survival rate of the spores are greatly decreased, and a using effect of the bio - control fungus is seriously reduced. Therefore, providing a culture medium for increasing a growth rate and stress resistance of the parasitical fungus Esteya vermicola in the Bursaphelenchus xylophilus and a preparation method of the culture medium is a problem that urgently needs to be solved by those skilled in the art.
    Summary In view of this, the present invention provides a culture medium for increasing a growth rate and stress resistance of a parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus and a preparation method of the culture medium, for shortening a growth cycle of the Esteya vermicola, increasing spore production of the Esteya vermicola, particularly increasing a ratio of crescent spores capable of adhering and causing death of Bursaphelenchus xylophilus, and increasing drought stress and ultraviolet stress resistance of the spores in a natural environment.
    To realize the above purposes, technical solutions of the present invention are adopted as follows: The culture medium for increasing the growth rate and stress resistance of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus specifically includes the following components: 170 - 230 g/L of potatoes, 15 - 25 g/L of glucose, 17 - 23 g/L of agar, 0.05 - 0.15 g/L of glycine and 4 - 6 g/L of calcium chloride. A pH value of the culture medium is 6 - 9.
    Further, the culture medium for increasing the growth rate and stress resistance of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus specifically includes the following components: 200 g/L of potatoes, 20 g/L of glucose, 20 g/L of agar, 0.1 g/L of glycine and 5 g/L of calcium chloride. A pH value of the culture medium is 6 - 9.
    Further, the potatoes are peeled potatoes.
    Further, the pH value is 7.
    Further, the preparation method of the culture medium for increasing the growth rate and stress resistance of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus specifically includes the following steps:
    (1) peeling and chopping potatoes, adding water for boiling, performing gauze filtration, and collecting filtrate; (2) adding glucose, agar, glycine and calcium chloride, adding distilled water, regulating a pH value, and fixing the volume; and (3) performing autoclaved sterilization on a solution with the constant volume at 121°C and
    1.05x105 Pa for 20 min, thereby obtaining the culture medium. Further, in the step (2), the water is added for bailing for 30 min. Further, an application of the culture medium for increasing the growth rate and stress resistance of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus in culture of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus specifically includes inoculating the Esteya vermicola to the culture medium for culture, wherein a culture temperature is 23 - 31°C, and culture time is 7 - 14 days. Further, the culture temperature is 26°C, and the culture time is 7 days. Through the above technical solutions, compared with the prior art, the present invention discloses and provides the culture medium for increasing the growth rate and stress resistance of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus and the preparation method of the culture medium. The culture medium increases the growth rate of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus, shortens the culture time, increases the spore production of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus, particularly obviously increases the ratio of the crescent spores capable of adhering and causing death of Bursaphelenchus xylophilus, increases the drought stress and ultraviolet stress resistance of the spores in the natural environment, and provides a favorable condition for use of the parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus. Detailed Description The technical solutions in the embodiments of the present invention will be clearly and fully described below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. Embodiment 1 A preparation method of a culture medium for increasing a growth rate and stress resistance of a parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus specifically includes the following steps: (1) potatoes were peeled and chopped, 1 L of water was added, water was boiled for 30 min, gauze filtration was performed, and filtrate was collected;
    (2) 20 g of glucose, 20 g of agar, 0.1 g of glycine and 5 g of calcium chloride were added, distilled water was added, a pH value was regulated to 7, and the volume was fixed to 1 L; and (3) a solution with the constant volume was subjected to autoclaved sterilization at 121°C and
    1.05% 10° Pa for 20 min, thereby obtaining the culture medium.
    The culture medium was poured into a culture dish under a sterile condition; after the culture medium in the culture dish was solidified, sterilized cellophane paper of which the diameter is slightly smaller than the culture dish is covered; then an activated parasitical fungus Esteya vermicola in Bursaphelenchus xylophilus was transferred onto the culture medium covered by the cellophane paper, and 10 plates were inoculated; and after inoculation, dark culture was performed in an incubator at 26°C.
    Within 15 days after culture, a diameter of a colony was measured by a ruler and counted; the fungus colony was scraped off from the solid medium covered by the cellophane paper; lots of the colonies were collected and divided into two parts, and each part was placed on 5 plates; the colonies on the 5 plates were placed in a drying oven at 80°C, continuously dried for two days, and weighed, and dry weight of the fungus colonies was recorded; colonies on the other 5 plates were transferred into a 50 ml of centrifuge tube, 0.5% of Tween 80 was added, and several glass beads were added; and the colonies were oscillated on an oscillator, and spore production and a ratio of crescent spores were counted by a blood counting chamber.
    Reference Example 1 Referring to the preparation method and counting method of the culture medium in Embodiment 1, 100 ug/ml of serine, 100 ug/ml of L - methionine and 100 pg/ml of L - leucine were respectively added into another 3 groups of culture media; a culture medium without adding any amino acid was taken as a control group; and repeated trial treatment was performed in each group for 3 times.
    The diameter of the colonies, the dry weight of the colonies, the spore production and the ratio of the crescent spores accounting for the total spores in Embodiment 1 and Reference Example 1 are detected, and results are as shown in Table 1.
    Table 1 Fungal Growth and Spore Production Conditions in Culture Media When Different Amino Acids Are Added _ Treatment Diameter ~~ Dryweight Spore ~~ Ratioof group (cm) (mg) production crescent (10° CFU/g) spores (%) "Control 23240242 133%11° 404¥020° 50.144#355° Serine 2.610.178 37.54£2.5° 4.01+0.31° 68.99+3.33°
    L- 2.70+0.16 48.142.5° 5.02+0.40° 72.55+3.19P methionine L - leucine 2.79+0.21" 56.813.5° 4.50+0.23% 75.2314.09° Glycine 3.02+0.19° 58.6+3.7° 6.96+0.48° 82.17+4.341 Notes: numerical values in the table are mean value + standard error (n=3), and letters at the back upper part of data in the same line represent significant differences on 0.05 level.
    Embodiment 2 5 According to the preparation method of the culture medium in Embodiment 1 and Reference Example 1, 100 ug/ml of glycine, 100 ug/ml of serine, 100 ug/ml of L - methionine and 100 pg/ml of L - leucine were respectively added into culture media; a culture medium without adding any amino acid was taken as a control group; after fungus inoculation, 200 pl of about 2000 pieces of Bursaphelenchus xylophilus was added onto a colony growing for 8 days; crescent spores of the fungus Esteya vermicola adhered to body surfaces of the Bursaphelenchus xylophilus, mainly the head and the tail adhered, the body surfaces and muscle layers of the Bursaphelenchus xylophilus were punctured, nutrients were absorbed, mycelia germinated to produce spores, and finally the nutrients of the Bursaphelenchus xylophilus were depleted to make the Bursaphelenchus xylophilus die; observation was performed under a microscope within 10 hours, and an adherence rate of the Bursaphelenchus xylophilus was counted; observation was performed within 28 hours, and a lethality rate of the Bursaphelenchus xylophilus was counted.
    Results are as shown in Table 2. Repeated trial treatment was performed in each group for 3 times.
    Table 2 Adherence Rate and Lethality Rate of Bursaphelenchus xylophilus in Culture Media When Different Amino Acids Are Added “Treatment group Adherence rate (%) Lethality rate (%) “Control 70.75%325° 7360x390° Serine 92.21+4.70° 84.54+4.00 L - methionine 91.20+4.15P 88.45+3.15M L - leucine 90.35+3.85" 89.97+4.10" Glycine 97.65+5.10° 94.7514.85° Notes: numerical values in the table are mean value + standard error (n=3), and letters at the back upper part of data in the same line represent significant differences on 0.05 level.
    Embodiment 3 According to the preparation method of the culture medium in Embodiment 1 and Reference Example 1, 100 ug/ml of glycine, 100 pg/ml of serine, 100 ug/ml of L - methionine and 100 pg/ml of L - leucine were respectively added into culture media; a culture medium without adding any amino acid was taken as a control group; after fungus inoculation for 8 days, spores of Esfeya vermicola colonies were collected; 200 ul of collected suspension of spores cultured by different amino acid culture media was uniformly added into the bottom of a culture dish; exposure was respectively performed at 25°C for 0.5 hour, 1 hour, 8 hours and 24 hours; then 1 ml of sterile water was added, and the spores were stirred by a glass rod; and 200 pl of spore suspension was absorbed and added onto water agar; and within 24 hours after culture, a germination rate of the spores was detected.
    The fungus Esteya vermicola was transferred into culture media added with four different amino acids; within 8 days after culture, spores of Esteya vermicola colonies were collected; the Esteya vermicola spores were coated on a water - agar medium; the medium coated with the spores was placed under an ultraviolet lamp G40T10 to be respectively irradiated for 2 minutes, 4 minutes, 6 minutes and 8 minutes, and a medium without ultraviolet radiation was taken as a control group; the culture medium subjected to ultraviolet radiation treatment was placed in an incubator for performing dark culture at 26°C for 24 hours, a germination rate of the spores was counted, and results were as shown in Table 3; and repeated trial treatment was performed in each group for 3 times.
    Table 3 Germination rate of Spores in Culture Media When Different Amino Acids Are Added “Treatme Treatm 0 Germinationrate%) nt enttime Control Serine ~~ L- L-leucine Glycine manner (min) methionine 30 713x066° 11.1840.539 13.18+0.45° 15.1:069° 19.67+127° 60 5.51+0.432 7.02+0.23P 7.80+0.23P 9.36+0.45° 13.04+0.48¢ Drought 480 4.51+0.17° 6.17+0.50 6.77+0.21° 7.88+0.42° 10.81+0.34¢ 1440 4.02+0.31° 5.6910.14° 5.9310.45° 6.07+0.31° 8.7510.621 2 3551£1.09° 41.00+1.43P 42.55+41.150 45.841+1.75% 44011163 Ultraviol 4 3.97+0.19° 5.42+0.08 7.91+0.17° 9.16+0.80° 11.90+0.72° et 6 2.03+0.05° 2.17+0.16° 2.83+0.18° 4.37+0.53P 5.39+0.50 radiation 8 0.90+0.13° 1.25+0.20° 1.65+0.08° 2.17+0.23P 2.25+0.16° Notes: numerical values in the table are mean value + standard error (n=3), and letters at the back upper part of data in the same line represent significant differences on 0.05 level.
    The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications made to these embodiments will be apparent to those skilled in the art. General principles defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein but will conform to the widest scope consistent with the principles and novel features disclosed herein.
    权利要求:
    Claims (8)
    [1]
    A culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus, comprising in particular the following ingredients: 170 - 230 g/L potatoes, 15 - 25 g/L glucose, 17 - 23 g/L agar, 0.05 - 0.15 g/L glycine and 4 - 6 g/L calcium chloride, the pH value being 6 - 9.
    [2]
    The culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to claim 1, wherein the culture medium comprises in particular the following ingredients: 200 g/L potatoes, 20 g/L glucose, 20 g/L agar, 0.1 g/L glycine and 5 g/L calcium chloride.
    [3]
    The culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to claim 1 or 2, wherein the potatoes are peeled potatoes.
    [4]
    The culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to claim 1 or 2, wherein the pH value is 7.
    [5]
    A culture medium preparation method for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to any one of claims 1 to 4, which method comprises in particular the following steps: (1) peeling and cutting potatoes into pieces, adding water for cooking, performing gauze filtration and collecting filtrate; (2) adding glucose, agar, glycine and calcium chloride, adding distilled water, adjusting the pH and recording the volume; and (3) performing autoclaved sterilization on a constant volume solution at 121°C and 1.05 x 10° Pa for 20 minutes to obtain the culture medium.
    [6]
    6. The culture medium preparation method for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in
    Bursaphelenchus xylophilus according to claim 5, wherein in step (2), water is added to boil for 30 minutes.
    [7]
    A use of the culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to any one of claims 1 to 6 in the culture of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus, wherein the Esteya vermicola is inoculated into the culture medium for the culture; the culture temperature is 23-31°C; and the culture time is 7-14 days.
    [8]
    The use of the culture medium for increasing the growth rate and stress resistance of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus in the culture of the parasitic fungus Esteya vermicola in Bursaphelenchus xylophilus according to claim 7, wherein the culture temperature is 26°C, and the culture time is 7 days.
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    同族专利:
    公开号 | 公开日
    CN111172093A|2020-05-19|
    NL2025554B1|2021-12-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    AU2015201322B2|2008-04-07|2016-08-25|Basf Corporation|Stable aqueous spore-containing formulation|
    CN109370949B|2018-11-29|2022-02-08|成昌根|Method for rapidly applying Islamic irasciaensis solid culture product to prevent and treat pine wilt|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN202010028143.9A|CN111172093A|2020-01-10|2020-01-10|Culture medium for improving growth speed and stress resistance of Esteya vermicola and preparation method thereof|
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