CN108676734B - Entomopathogenic fungi and application thereof - Google Patents

Entomopathogenic fungi and application thereof Download PDF

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CN108676734B
CN108676734B CN201811021179.3A CN201811021179A CN108676734B CN 108676734 B CN108676734 B CN 108676734B CN 201811021179 A CN201811021179 A CN 201811021179A CN 108676734 B CN108676734 B CN 108676734B
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beauveria bassiana
potato
entomopathogenic fungus
early blight
hubb
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CN108676734A (en
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丁俊男
于少鹏
胡宝忠
来永才
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Harbin University
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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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    • C12N1/145—Fungal isolates
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    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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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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Abstract

The invention discloses an entomopathogenic fungus and application thereof, and relates to an entomopathogenic fungus and application thereof. The invention provides an entomopathogenic fungus and application thereof. The entomopathogenic fungus is beauveria bassiana HUBB.No. 08, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, the preservation date is 6 and 11 days in 2018, and the preservation number is CGMCC No. 15866. The invention is applied to the prevention and control of potato diseases. The strain has strong capability of inhibiting the growth of hyphae of potato early blight bacteria, increases along with the increase of volume concentration, has long lasting period and obvious bacteriostatic effect, and has bacteriostatic efficiency of 81.73 percent, thereby showing that the strain HUBB.No. 08 has good antagonistic action on the potato early blight. The invention is applied to the technical field of environmental microorganisms.

Description

Entomopathogenic fungi and application thereof
Technical Field
The invention relates to an entomopathogenic fungus and application thereof.
Background
Potato Early Blight (Potato Early Blight) is a disease causing serious damage to potatoes caused by Alternaria solani (el. et Mart.) Jones et Grout, and is commonly occurring worldwide, and is considered as the second largest Potato disease second only to Potato late Blight in developing countries. Early blight causes large area of reduction in yield of potatoes, and brings serious economic loss. The disease occurs in different degrees in potato production areas in China, and is in an increasing trend in recent years, and the harm degree of local areas is not inferior to that of the potato late blight. The pathogenic bacteria of the potato early blight not only harm potatoes, but also infect tomato, eggplant, hot pepper, beet, mustard and other crops to cause the early blight. The potato early blight is widely occurred in the potato planting areas in the world and is an important potato disease in the world. In recent years, early blight frequently occurs in a plurality of potato production areas and has a rising trend, the yield of potatoes can be reduced, and the yield can be reduced by more than 50% in severe years.
Disclosure of Invention
The invention provides an entomopathogenic fungus and application thereof.
The entomopathogenic fungus is Beauveria bassiana (Beauveria bassiana) HUBB.No. 08, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo Beichen, the preservation date is 2018, 6 and 11 days, and the preservation number is CGMCC No. 15866.
The application of the entomopathogenic fungi is the application in preventing and controlling potato diseases.
The invention obtains Beauveria bassiana (Beauveria bassiana) HUBB.No. 08 according to the following modes:
separating entomopathogenic fungi primary type fungi from soil samples in Harbin city by using a tenebrio molitor trapping method:
infecting yellow mealworm (5-instar larva) with the separated entomopathogenic fungi, soaking the dead larva in 75% alcohol for 5s, scraping a small amount of white spore from the larva under aseptic condition on PDA solid culture medium, culturing in 28 deg.C biochemical incubator for 2-3 days, selecting white colony as birth type colony, and inoculating in improved Martin culture solution (filtered potato cooking solution 1000mL, glucose 20g, KH)2PO4 3g、MgSO4·7H2O1.5 g and vitamin B18 mg), carrying out shake culture at the temperature of 28 ℃ for 24h at 180r/min, transferring the mixture into a fermentation culture solution according to the volume ratio of 1%, carrying out shake culture fermentation for 36-48h, carrying out refrigerated centrifugation in a centrifuge tube, and taking supernatant to obtain sterile fermentation liquor.
The color of the colony observed by the separated and screened entomopathogenic fungi on a nutrient medium under an optical microscope is milk white. The colony is powdery, thin, wrinkled and radially grown. The bottom of the colony is colorless or yellowish. The spore-forming cells of the strain are recurrent or single, conidiophores are repeatedly sporulated towards the top axis, and as a result, a knee-shaped bent sporulation axis with small dentations is formed. The conidiophores are smooth and spike-shaped, the conidiophores are transparent, the walls are thin and spherical, and the surfaces are smooth.
The molecular identification of the entomopathogenic fungi obtained by separation and screening is carried out according to the following steps of extracting the total DNA of the strain and carrying out PCR amplification by taking genome DNA as a template according to the general primers of the fungi. And then recovering and purifying the PCR product by using a gel recovery kit, and then cloning, transforming and sequencing. The homology comparison of the sequencing result and a 16S rDNA sequence in Genbank shows that the homology of the HUBB.No. 08 strain is the highest with that of beauveria, and is more than 99 percent. The strain HUBB.No. 08 is determined to be Beauveria bassiana (Beauveria bassiana) by combining the morphological characteristics of thalli and the molecular identification result, and is finally named as the Beauveria bassiana (Beauveria bassiana) HUBB.No. 08.
Beauveria bassiana (B.bassiana) is one of the insecticidal fungi widely used for biological control of pests at home and abroad, can effectively control the population quantity of pests, and does not harm other natural enemy insects and beneficial organisms at the same time.
The invention effectively prevents and treats the potato early blight by using a biological prevention and treatment means, is more environment-friendly compared with other traditional chemical prevention and treatment means, has no pesticide residue, and does not generate drug resistance.
Drawings
FIG. 1 is a photograph of a beauveria bassiana HUBB.No. 08 sterile fermentation broth on a modified Martin medium for 3 days when producing an zone of inhibition on potato early blight bacteria;
FIG. 2 is a photograph of a Beauveria bassiana HUBB.No. 08 sterile fermentation broth for 7 days on a modified Martin medium to produce an zone of inhibition on potato early blight bacteria.
Detailed Description
The first embodiment is as follows: the entomopathogenic fungus is Beauveria bassiana (Beauveria bassiana) HUBB.No. 08, which is preserved in the common microorganism center of China general microbiological culture Collection center, the preservation address is No. 3 of No.1 Siro-Lu-1 of Beijing market and Chaoyang district, the preservation date is 2018, 6 and 11 days, and the preservation number is CGMCC No. 15866.
The embodiment is that the Beauveria bassiana (Beauveria bassiana) HUBB.No. 08 is obtained according to the following mode:
separating entomopathogenic fungi primary type fungi from soil samples in Harbin city by using a tenebrio molitor trapping method:
infecting yellow mealworm (5-instar larva) with the separated entomopathogenic fungi, soaking the dead larva in 75% alcohol for 5s, scraping a small amount of white spore from the larva under aseptic condition on PDA solid culture medium, culturing in 28 deg.C biochemical incubator for 2-3 days, selecting white colony as birth type colony, and inoculating in improved Martin culture solution (filtered potato cooking solution 1000mL, glucose 20g, KH)2PO4 3g、MgSO4·7H2O1.5 g and vitamin B18 mg), carrying out shake culture at the temperature of 28 ℃ for 24h at 180r/min, transferring the mixture into a fermentation culture solution according to the volume ratio of 1%, carrying out shake culture fermentation for 36-48h, carrying out refrigerated centrifugation in a centrifuge tube, and taking supernatant to obtain sterile fermentation liquor.
The color of the colony observed by the separated and screened entomopathogenic fungi on a nutrient medium under an optical microscope is milk white. The colony is powdery, thin, wrinkled and radially grown. The bottom of the colony is colorless or yellowish. The spore-forming cells of the strain are recurrent or single, conidiophores are repeatedly sporulated towards the top axis, and as a result, a knee-shaped bent sporulation axis with small dentations is formed. The conidiophores are smooth and spike-shaped, the conidiophores are transparent, the walls are thin and spherical, and the surfaces are smooth.
The molecular identification of the entomopathogenic fungi obtained by separation and screening is carried out according to the following steps of extracting the total DNA of the strain and carrying out PCR amplification by taking genome DNA as a template according to the general primers of the fungi. And then recovering and purifying the PCR product by using a gel recovery kit, and then cloning, transforming and sequencing. The homology comparison of the sequencing result and a 16S rDNA sequence in Genbank shows that the homology of the HUBB.No. 08 strain is the highest with that of beauveria, and is more than 99 percent. The strain HUBB.No. 08 is determined to be Beauveria bassiana (Beauveria bassiana) by combining the morphological characteristics of thalli and the molecular identification result, and is finally named as the Beauveria bassiana (Beauveria bassiana) HUBB.No. 08.
Beauveria bassiana (B.basisana) is one of the insecticidal fungi widely used for biological control of pests at home and abroad, can effectively control the population quantity of pests, and does not damage other natural enemy insects and beneficial organisms.
The second embodiment is as follows: the application of the entomopathogenic fungi in the embodiment is the application in potato disease control.
The method effectively prevents and treats the potato early blight by using a biological prevention and treatment means, is more environment-friendly compared with other traditional chemical prevention and treatment methods, has no pesticide residue, and does not generate drug resistance.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the entomopathogenic fungi are applied to inhibiting potato early blight bacteria. The rest is the same as the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the first or second embodiment in that: the potato early blight is prevented and controlled by adopting the beauveria bassiana bacterial liquid capable of inhibiting the potato early blight. The other embodiments are the same as the second or third embodiment.
The fifth concrete implementation mode: this embodiment is different from one of the second to fourth embodiments in that: the preparation method of the beauveria bassiana bacterial liquid capable of inhibiting potato early blight comprises the following steps: beauveria bassiana (Beauveria bassiana) HUBB.No. 08 is inoculated in a liquid culture medium, shaking, fermenting and culturing are carried out for 36-48h under the condition of 28 ℃ and 180r/min, the obtained product is poured into a centrifuge tube for freezing and centrifuging, and supernatant is taken to obtain bacterial liquid. The other is the same as one of the second to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the second to fifth embodiments in that: the liquid culture medium is an improved Martin culture medium, and the formula is as follows: 1L of modified Martin's medium is prepared from 1000mL of filtered potato cooking liquor, 20g of glucose, 3g of KH2PO41.5g of MgSO 24·7H2O and 8mg of vitamin B1. The rest is the same as one of the second to fifth embodiments.
The seventh embodiment: this embodiment is obtained from Beauveria bassiana (Beauveria bassiana) HUBB.No. 08 in the following manner:
separating entomopathogenic fungi primary type fungi from soil samples in Harbin city by using a tenebrio molitor trapping method:
infecting yellow mealworm (5-instar larva) with the separated entomopathogenic fungi, soaking the dead larva in 75% alcohol for 5s, scraping a small amount of white spore from the larva under aseptic condition on PDA solid culture medium, culturing in 28 deg.C biochemical incubator for 2-3 days, selecting white colony as birth type colony, and inoculating in improved Martin culture solution (filtered potato cooking solution 1000mL, glucose 20g, KH)2PO4 3g、MgSO4·7H2O1.5 g and vitamin B18 mg), carrying out shaking culture at the temperature of 28 ℃ for 24 hours at the speed of 180r/min, transferring the mixture into a fermentation culture solution according to the volume ratio of 1%, carrying out shaking culture and fermentation for 36-48 hours, freezing and centrifuging, and taking supernatant fluid to obtain sterile fermentation liquor.
The specific implementation mode is eight: the identification of Beauveria bassiana HUBB.No. 08 of the embodiment:
the color of the colony observed by the separated and screened entomopathogenic fungi on a nutrient medium under an optical microscope is milk white. The colony is powdery, thin, wrinkled and radially grown. The bottom of the colony is colorless or yellowish. The spore-forming cells of the strain are recurrent or single, conidiophores are repeatedly sporulated towards the top axis, and as a result, a knee-shaped bent sporulation axis with small dentations is formed. The conidiophores are smooth and spike-shaped, the conidiophores are transparent, the walls are thin and spherical, and the surfaces are smooth.
The molecular identification of the entomopathogenic fungi obtained by separation and screening is carried out according to the following steps of extracting the total DNA of the strain and carrying out PCR amplification by taking genome DNA as a template according to the general primers of the fungi. And then recovering and purifying the PCR product by using a gel recovery kit, and then cloning, transforming and sequencing. The DNA sequence is shown as SEQ ID NO: 1 is shown. The homology comparison of the sequencing result and a 16S rDNA sequence in Genbank shows that the homology of the HUBB.No. 08 strain is the highest with that of beauveria, and is more than 99 percent. The strain HUBB.No. 08 is determined to be Beauveria bassiana (Beauveria bassiana) by combining the morphological characteristics of thalli and the molecular identification result, and is finally named as the Beauveria bassiana (Beauveria bassiana) HUBB.No. 08.
The functional verification of Beauveria bassiana HUBB.No. 08 of the embodiment on the inhibition of potato early blight bacteria is as follows:
test 1:
determination of antagonism of sterile fermentation liquor to potato early blight by using gradient dilution method
1. Early preparation: the potato early blight bacteria stored in the laboratory are inoculated on a potato glucose agar medium (PDA) and placed in a constant temperature incubator at 26 ℃, and the cultured hyphae grow over the culture dish for later use.
2. Preparation of potato dextrose agar medium (PDA): the method comprises the steps of firstly cleaning and peeling potatoes, weighing 200g of the potatoes, cutting the potatoes into small pieces, adding water, boiling the small pieces thoroughly (boiling for 20-30 minutes and being punctured by a glass rod), filtering the small pieces with eight layers of gauze, heating, adding 15-20g of agar according to actual experiments, continuously heating, stirring and uniformly mixing, adding glucose after the agar is dissolved, stirring uniformly, slightly cooling, supplementing water to 1000ml, subpackaging test tubes or conical bottles, plugging and binding, sterilizing at 115 ℃ for about 20 minutes, taking out the test tubes, swinging the test tubes to an inclined plane or shaking uniformly, cooling and storing for later use.
3. And (3) determination of antagonism: under aseptic conditions, a punch with an inner diameter of 5mm was used to punch a hole in PDA medium, 60. mu.L of the fermentation broth (named Bb08) was added to the hole, and a block of Alternaria solani was inoculated 4cm from the hole. With the addition of sterile water as a control (designated BEL), each treatment was repeated 3 times. And (5) placing the culture medium in a constant temperature incubator at 26 ℃, and measuring the width of the bacteriostatic band when the control bacterial colony grows to be full.
4. And (4) analyzing results: according to determination, the beauveria bassiana HUBB.No. 08 sterile fermentation liquid has an obvious inhibition effect on potato early blight, hypha bypasses a sterile fermentation liquid inoculation hole from the 1 st day of the generated inhibition zone to the 7 th day, the inhibition zone with the width of 5mm is averagely generated around antagonistic bacteria, the potato early blight close to one side of the antagonistic bacteria stops growing, and the antagonistic action result on the potato early blight is shown in figures 1 and 2, wherein figure 1 is a picture of the beauveria bassiana HUBB.No. 08 sterile fermentation liquid on an improved Martin medium when the inhibition zone is generated on the potato early blight for 3 days; FIG. 2 is a photograph of a Beauveria bassiana HUBB.No. 08 sterile fermentation broth for 7 days on a modified Martin medium to produce an zone of inhibition on potato early blight bacteria. As can be seen from the figures 1 and 2, the beauveria bassiana HUBB.No. 08 has obvious bacteriostatic effect on the potato early blight bacteria on the seventh day, which shows that the entomopathogenic fungus beauveria bassiana HUBB.No. 08 has longer lasting period for inhibiting the potato early blight bacteria.
The sterile fermentation liquid of the entomopathogenic fungus beauveria bassiana HUBB.No. 08 has obvious antagonistic action on potato early blight bacteria, has longer lasting period, can well inhibit the growth of the potato early blight bacteria close to one side of the antagonistic bacteria, has obvious antibacterial effect, and has the antibacterial efficiency of 81.73 percent.
Sequence listing
<110> Harbin college
<120> entomopathogenic fungus and application thereof
<160> 1
<210> 1
<211> 577
<212> DNA
<213> Beauveria bassiana (Beauveria bassiana)
<400> 1
tagtcctacg cgcgatcagg atgaaacgta agctccagtg caagtcttca acccacaaaa 60
tgccgcaccg gcgcagcccc agggccacgc tcgacataat gacgcgtctc cagcggcgcc 120
tgcccggcgg tgaggtaaag tcccggccgc cacacgacgg ccaggggttg cggctggagg 180
ggttcccctc cagctcccaa ctttactgcg agcttgtccg tacgggcggt cttacgaccg 240
cccgcgttac acgcaagttt ctaagctact aagtgaccta agacgttaag tgtaatgaat 300
agcgcaaagc gacgcaagaa gtagctacgg tctcggttct ctaggcaaca actttcaaaa 360
ctaagtaaac aaaacggaac gccgcataag tcttctacga ccttatgttc tcaaactcca 420
ggggccgccc ggcgaccagg tcaggcgcag gcccgacccc gctcaggcgg cttcgttgct 480
atccatccaa gtgtcttccc aatccctcaa cttttgagcc attactaggg aggcgaccaa 540
gtggttgcct ctggaacaat gctaaaaaat gaagtat 577

Claims (5)

1. An entomopathogenic fungus is characterized in that the entomopathogenic fungus is Beauveria bassiana (Beauveria bassiana) HUBB.No. 08, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, the preservation date is 6 months and 11 days in 2018, and the preservation number is CGMCC No. 15866.
2. The use of an entomopathogenic fungus according to claim 1 for inhibiting early blight of potato.
3. The use of an entomopathogenic fungus according to claim 2, characterized in that the potato early blight is controlled by using a bacterial solution of beauveria bassiana which inhibits potato early blight.
4. The application of the entomopathogenic fungus strain as claimed in claim 3, wherein the preparation method of the bacterial liquid of beauveria bassiana for inhibiting potato early blight comprises the following steps: beauveria bassiana (Beauveria bassiana) HUBB.No. 08 is inoculated in a liquid culture medium, shaking, fermenting and culturing are carried out for 36-48h under the condition of 28 ℃ and 180r/min, the obtained product is poured into a centrifuge tube for freezing and centrifuging, and supernatant is taken to obtain bacterial liquid.
5. The use of an entomopathogenic fungus according to claim 4, wherein the liquid medium is a modified Martin's medium having a formula of: 1L of modified Martin's medium is prepared from 1000mL of filtered potato cooking liquor, 20g of glucose, 3g of KH2PO41.5g of MgSO 24·7H2O and 8mg of vitamin B1.
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CN111662830A (en) * 2020-07-14 2020-09-15 哈尔滨学院 Entomopathogenic fungi for inhibiting wheat damping-off and preparation method thereof
CN114456949B (en) * 2022-01-13 2023-06-16 贵州民族大学 Beauveria bassiana JSHA-MD912 and application thereof
CN117431175B (en) * 2023-10-08 2024-05-24 云南师范大学 Aquatic Lei Fusen bacterium ST172 and application thereof

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CN105918330A (en) * 2016-05-24 2016-09-07 重庆大学 New application of sirolimus in inhibition of plant alternaria solani
CN107347922A (en) * 2017-08-18 2017-11-17 临沂市农业科学院 A kind of application of Strain of Beauveria bassiana

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