CN114480145B - Strain with strong pathogenicity to walnuts apocynum and application thereof - Google Patents
Strain with strong pathogenicity to walnuts apocynum and application thereof Download PDFInfo
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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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- C—CHEMISTRY; METALLURGY
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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
- C12N1/02—Separating microorganisms from their culture media
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to the technical field of biological control, and discloses a strain with strong pathogenicity on a walnut apocynum, which is beauveria bassiana and is preserved in China general microbiological culture collection center (CGMCC NO) at 9 and 23 of 2021, wherein the strain is prepared by the following steps: 23261. the beauveria bassiana strain is obtained by separating the beauveria bassiana strain from the stiff worms of the walnut long-foot elephant collected in forests for the first time, is simple to culture, quick in growth, large in spore yield and high in spore germination rate, has strong pathogenicity to the walnut long-foot elephant adults through experiments, is environment-friendly, is not easy to generate drug resistance, and can be widely applied to biological control of the walnut long-foot elephant.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to a strain with strong pathogenicity on walnuts, and application thereof.
Background
The walnut is one of important economic tree species in China, the wood, fruits, branches, leaves and shells of the walnut have high utilization and development values, the walnut industry is established as one of dominant industries of regional economy and new rural economy development in many areas, and the walnut industry can promote farmers to increase income, pull regional economy development, improve ecological environment and be popularized in multiple provinces in China. However, as new varieties are blindly introduced, the varieties are improved and the cultivation area is enlarged continuously, and fruit farmers relax and manage, so that the occurrence of walnut diseases and insect pests is serious, the walnut growth is one of important insect pests of the walnut insect pests, and the single plant yield is low and the quality is reduced.
The long-foot elephant (Alcidodesj. Juglans Chao) is also called walnut fruit weevil, walnut weevil, etc., belongs to coleoptera weevil family, is an important boring pest of walnut fruits, belongs to moderate dangerous forestry pests, and is distributed in walnut producing areas in China. The insect is most seriously damaged by the larvae, the larvae eat kernels in the walnut fruits, so that the fruits fall off, and the fruit trees are dead in severe cases, which is a main reason for influencing a large number of abnormal falling fruits of the walnut and reducing the yield; adults gnaw tender leaves, tender tips and young fruit peels to influence the growth of fruit trees, so that the yield is reduced.
At present, the prevention and treatment method for the walnut apocary mainly adopts the methods of manual fruit dropping, trunk injection, crown spraying and the like. However, the method is time-consuming and labor-consuming, causes food safety problems and causes serious environmental pollution, so that the research on an efficient, stable and pollution-free control method is needed to be solved.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide a beauveria bassiana strain with strong pathogenicity to the apocynum walnuts, wherein the strain is beauveria bassiana, and the strain is preserved in China general microbiological culture collection center (CGMCC) for 9 months 23 days in 2021, and the strain preservation number is CGMCCNO:23261 the preservation address is North Chen West Lu No.1 and No. 3 in the Chaoyang district of Beijing city.
The ITS sequence of the strain is shown as SEQ ID NO. 1.
The second object of the invention is to provide a specific isolation and purification method of the strain.
The beauveria bassiana strain is obtained by separating from stiff worms of the walnut apocynum venetum collected in walnut forests, immersing the walnut apocynum venetum in 70% ethanol for sterilization for 30s, then sterilizing for 1.5min by using 0.1% mercuric chloride, rinsing for 3 times by using sterilized water, sucking water on the adults of the walnut apocynum venetum by using sterilized filter paper, and then placing the adults in a PDA flat plate. Culturing in a constant temperature incubator at 27deg.C for 2-3d until mycelia grow around the insects, picking mycelia, transferring to a new PDA plate, repeating for 3 times, and separating single colony of germs by dilution purification method. And finally, storing the separated and purified strain on a PDA slant culture medium, and storing in a refrigerator at 4 ℃. The colony is white long velvet in the initial stage of culture, the back is colorless, and the mycelium is transparent when observed under an optical microscope; however, the colony is light yellow powder at the later stage of culture, the back of the colony is light yellow, hypha and spore-forming cells are obviously reduced, only the enlarged irregular vesicle hyperplasia is seen, a large number of spores are gathered into a sphere on the surface of the vesicle under a microscope, and the conidium is transparent and mostly in an oval shape with the diameter of 1.05 x 2.14-1.65 x 2.49 mu m.
The invention further aims to provide application of the strain in preventing and treating the walnut apostrophe. The specific application is that the beauveria bassiana strain is cultured to prepare spore liquid and spore-containing microbial inoculum, and the spore concentration of the strain is 1 multiplied by 10 when the strain is applied 6 spore/mL-1X 10 8 spores/mL to achieve green control of the walnut apocarya.
The invention has the beneficial effects that: the beauveria bassiana strain is obtained by separating the infected walnut apocynum, is simple to culture, rapid to grow, large in spore yield and high in spore germination rate for the first time, has strong pathogenicity to adults and nymphs of the walnut apocynum through experiments, is environment-friendly, is not easy to generate drug resistance, and can be widely applied to biological control of the walnut apocynum.
Drawings
FIG. 1 shows a colony formed by culturing a beauveria bassiana strain (with a preservation number of CGMCCNO: 23261) on a PDA plate, wherein A is the front surface of the colony, B is the back surface of the colony, and C, D is the morphological characteristics of hyphae;
FIG. 2 is an electron microscope scanning photograph of conidium and conidium peduncles of beauveria bassiana strain (preservation number is CGMCCNO: 23261);
FIG. 3 is an optical microscope photograph of conidium of beauveria bassiana strain (preservation number: CGMCCNO: 23261);
FIG. 4 is an electrophoresis chart of PCR amplification products (sample in the frame line) in the molecular characterization of the present invention.
Detailed Description
The present invention will be described in detail with reference to examples, which are not intended to be limiting.
PDA culture medium formula: peeled potato 200g, glucose 20g, agar 15-20g, distilled water 1000ml, natural pH.
The walnut apocarya is collected in Qingzhen city, guizhou province, and the plow is used for eight villages on the right.
Example 1: isolation and identification of beauveria bassiana
(1) Separating, purifying and preserving
Soaking the long-foot elephant of Juglandis in 70% ethanol, sterilizing for 30s, sterilizing with 0.1% mercuric chloride for 1.5min, rinsing with sterilized water for 3 times, sucking water on the adult of the long-foot elephant of Juglandis with sterilized filter paper, and placing in PDA plate. Culturing in a constant temperature incubator at 27deg.C for 2-3d until mycelia grow out around the insects, picking mycelia, transferring to a new PDA plate, repeating for 3 times, and separating single colony of bacteria by dilution purification to obtain rejuvenated beauveria bassiana strain with strong pathogenicity. The obtained high virulence strain was transferred to fresh PDA slant medium and then stored at 4 ℃. The strain is preserved in China general microbiological culture Collection center (CGMCCNO) of China general microbiological culture Collection center (China general microbiological culture collection center) for 9 and 23 of 2021: 23261 the preservation address is North Chen West Lu No.1 and No. 3 in the Chaoyang district of Beijing city.
(2) Identification of strains
Identification was performed based on the morphological characteristics of isolated strains, pure cultured colonies, hyphae and conidia. The experimental results show that: the strain obtained by separating from the adult insect body of the walnut apocarya naturally infected by the entomogenous fungi is subjected to rejuvenation, separation and purification on the strain according to the Koch rule by obtaining pure culture on a PDA culture medium, and the pure culture of the strain is obtained by separating, namely the beauveria bassiana strain. The colony characteristics are as follows: the colony is white long velvet in the initial stage of culture, the back is colorless, and the mycelium is transparent when observed under an optical microscope; however, until the later stage of culture, the colony is light yellow powder, the back of the colony is light yellow, hyphae and spore-forming cells are obviously reduced, only the enlarged irregular vesicle hyperplasia is seen, a large number of spores are gathered into a sphere on the surface of the vesicle under a microscope, and the conidium is transparent and mostly in an oval shape, and has a diameter of 1.05 x 2.14-1.65 x 2.49 mu m, as shown in the accompanying drawings of 1-3. According to the infection symptoms, colony and thallus characteristics of the walnut apocarya, the strain is analyzed to be beauveria bassiana.
Example 2 molecular characterization of strains
(1) Fungal genomic DNA extraction
Extracting genome DNA of beauveria bassiana strain by using a TSINGKE plant DNA extraction kit (general type), wherein the specific steps are as follows:
s1: placing spinCoulumn in a collectionTube, adding 250 μLBuferBL, and centrifuging at 12000rpm/min for 1min to activate the silica gel film;
s2: adding liquid nitrogen into a sample dry tissue (less than or equal to 20 mg), fully grinding, placing into a 1.5ml centrifuge tube after grinding, adding 400 mu LBufergP 1, vortex oscillating for 1min, and carrying out water bath at 65 ℃ for 10-30min, and taking out, reversing and uniformly mixing for full cracking;
s3: adding 150 mu LBufergP 2, vortex oscillating for 1min, and ice water bath for 5min;
s4: centrifuging at 12000rpm/min for 5min, and transferring the supernatant to a new centrifuge tube;
s5: adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, completely transferring the liquid into Spin coulurn, centrifuging at 12000rpm/min for 30s, and discarding the waste liquid;
s6: 500 μLBuferPw (absolute ethanol was added before use) was added to spinCoulumn, centrifuged at 12000rpm/min for 30s, and the waste liquid was discarded;
s7: 500 mu LWAshBuffer (absolute ethanol is added before use) is added into the spinCoulumn, the mixture is centrifuged at 12000rpm/min for 30s, and the waste liquid is discarded;
s8: repeating the operation step S7;
s9: putting spinCoulumn back into the collectionTube, centrifuging at 12000rpm/min for 2min, uncovering and airing for 1min;
s10: taking out spinCoulumn, placing into a clean centrifuge tube, adding 50-100 μLTEBuffer (preheated TEBuffer at 65deg.C) at the center of the adsorption film, standing at 20-25deg.C for 2min, and centrifuging at 12000rpm/min for 2min.
(2) PCR amplification
The ITS region universal primers for ribosomal DNA were purchased from TsingKe and the primer series were: ITS15'-TCCGTAGGTGAACCTGCGG-3', ITS45'-TCCTCCGCTTATTGATATGC-3'.
The extracted DNA sample is diluted with a proper amount and is used as a PCR template, amplification is carried out by using 1 XTSE 101 gold plate mix of the Optimaceae, and the components of an amplification system are as follows:
| 1×TSE101 gold medal mix | 45μL |
| ITS1(10p) | 2μL |
| ITS4(10p) | 2μL |
| DNA template | 1μL |
The above amplification system was amplified according to the following amplification procedure:
the amplified PCR products were subjected to agarose gel electrophoresis (2. Mu.L of sample+6. Mu.L of bromophenol blue) at 300V for 12min to obtain an identification gel (see FIG. 4).
(3) The PCR products after molecular sequencing and strain identification recovery were sequenced by Beijing qing biological technology Co. Splicing sequencing results by Contigexpress, removing inaccurate parts at two ends, comparing spliced sequences in NCBI database, and carrying out molecular identification of strain with similarity of 99% or above as standard.
The research results show that: and (3) taking the obtained fungus genome DNA as a template, carrying out PCR amplification by using specific primers ITS1 and ITS4 to obtain an amplification product, recovering and sequencing the PCR amplification product, wherein the result shows that the homology of the PCR amplification product with beauveria bassiana strain Beauveria bassiana in NCBI is 99%, and identifying the strain as beauveria bassiana.
The nucleotide sequence of the ITS sequence of the strain is:
5’-AACCTGCGGAGGGATCATTACCGAGTTTTCAACTCCCTAACCCTTCTGTGAACCTACCT TATCGTTGCTTCGGCGGACTCGCCCCAGCCCGGACGCGGACTGGACCAGCGGCCCGCCG GGGACCTCAAACTCTTGTATTCCAGCATCTTCTGAATACGCCGCAAGGCAAAACAAATGA ATCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATG CGATAAGTAATGTGAATTGCAGAATCCAGTGAATCATCGAATCTTTGAACGCACATTGCGC CCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACCTCCCCTG GGGGAGGTCGGCGTTGGGGACCGGCAGCACACCGCCGGCCCTGAAATGGAGTGGCGGC CCGTCCGCGGCGACCTCTGCGTAGTAATACAGCTCGCACCGGAACCCCGACGCGGCCAC GCCGTAAAACACCCAACTTCTGAACGTTGACCTCGAATCAGGTAGGACTACCCGCTGAA CTTAAGCATATCAATAAGGCGGAGGA-3’
example 3 virulence test of strains
(1) Preparation of fungal spore suspensions
Inoculating the preserved beauveria bassiana strain (CGMCCNO: 23261) into PDA culture medium, culturing at 28deg.C for 10 days, scraping conidium, and diluting with sterile water to 1×10 8 spores/mL, 1×10 7 spores/mL, 1×10 6 spores/mL, 1×10 5 spores/mL, 1×10 4 spore/mL spore suspension for use.
(2) Indoor toxicity measurement
The toxicity test was performed by the immersion method. Picking about 30 healthy and strong adults of the walnut apocarya at each treatment concentration (about 10 per repetition and 3 times in total), soaking the healthy and strong walnut apocarya in the suspension for 8s by adopting spore suspension with corresponding concentration, culturing fresh walnut branch leaves and walnut fruits which are cut, placing the branches and the walnut fruits in a glass bottle, sealing the top surface by a gauze, culturing at the temperature of 25 ℃, controlling the relative humidity to 60%, keeping the illumination period in darkness for 12 hours and lighting for 12 hours, and comparing the healthy and strong adults of the walnut apocarya treated by spraying distilled water (about 10 per repetition and 3 times in total), and observing and recording the death condition every day.
(3) Results
After the beauveria bassiana strain (CGMCCNO: 23261) is sprayed, the adults of the walnut long-foot image of each treatment begin to die 1 multiplied by 10 after 3 days of treatment 8 The mortality rate of the spores/mL concentration treated on day 7 is about 50%, the mortality rate reaches 80% on day 12, and a large number of mycelia and conidia grow out of joint parts of the insect bodies, and the mortality rate reaches 93.33% on day 25. 1X 10 4 spores/mL, 1×10 5 spores/mL, 1×10 6 spores/mL, 1×10 7 spores/mL, 1×10 8 The cumulative mortality of the spore/mL concentration treated walnuts apocynum imatum adults on the 25 th day after treatment was 56.67%, 66.67%, 80%, 83.33% and 93.33% respectively, and the cumulative mortality of the control treated walnuts apocynum imatum adults on the 25 th day after treatment was only 10%. The above 1×10 4 spores/mL, 1×10 5 spores/mL, 1×10 6 spores/mL, 1×10 7 spores/mL, 1×10 8 Spores/mThe cumulative mortality of L-concentration treated Juglandis apocaria at 25 days after treatment was significantly higher than that of the control, 1×10 6 spores/mL, 1×10 7 spores/mL, 1×10 8 The cumulative mortality of the walnut apocarya treated by the spore/mL concentration is higher than 80% at the 25 th day after the treatment, which shows that the walnut apocarya has remarkable killing effect on the walnut apocarya.
Therefore, the beauveria bassiana strain has a good killing effect on the apophyseal of the walnut, and can play an important role in biological control of the apophyseal of the walnut.
TABLE 1
Sequence listing
<110> institute of walnut in Guizhou province
<120> A strain having strong pathogenicity against Juglandis Manta elephant and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 561
<212> DNA
<213> Beauveria bassiana Strain (Beauvenia bassiana)
<400> 1
aacctgcgga gggatcatta ccgagttttc aactccctaa cccttctgtg aacctacctt 60
atcgttgctt cggcggactc gccccagccc ggacgcggac tggaccagcg gcccgccggg 120
gacctcaaac tcttgtattc cagcatcttc tgaatacgcc gcaaggcaaa acaaatgaat 180
caaaactttc aacaacggat ctcttggctc tggcatcgat gaagaacgca gcgaaatgcg 240
ataagtaatg tgaattgcag aatccagtga atcatcgaat ctttgaacgc acattgcgcc 300
cgccagcatt ctggcgggca tgcctgttcg agcgtcattt caaccctcga cctcccctgg 360
gggaggtcgg cgttggggac cggcagcaca ccgccggccc tgaaatggag tggcggcccg 420
tccgcggcga cctctgcgta gtaatacagc tcgcaccgga accccgacgc ggccacgccg 480
taaaacaccc aacttctgaa cgttgacctc gaatcaggta ggactacccg ctgaacttaa 540
gcatatcaat aaggcggagg a 561
Claims (5)
1. The strain with strong pathogenicity to the walnuts is characterized in that the strain is beauveria bassiana and is preserved in China general microbiological culture Collection center (CGMCC) in the year 2021, 9 and 23, and the preservation number is CGMCC NO:23261.
2. the use of the strain according to claim 1 for controlling walnuts.
3. Use according to claim 2, characterized in that the strain is cultivated to a spore fluid or a spore-containing bacterial agent.
4. Use according to claim 3, characterized in that the spore concentration of the strain at the time of administration is 1 x 10 6 spore/mL-1X 10 8 spores/mL.
5. An insecticide for preventing and treating walnut apocaria, which is characterized in that: a strain comprising the strain of claim 1.
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