CN116606742A - Penicillium oxalate MS01 and application thereof - Google Patents

Penicillium oxalate MS01 and application thereof Download PDF

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CN116606742A
CN116606742A CN202310450434.0A CN202310450434A CN116606742A CN 116606742 A CN116606742 A CN 116606742A CN 202310450434 A CN202310450434 A CN 202310450434A CN 116606742 A CN116606742 A CN 116606742A
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penicillium
prodenia litura
penicillium oxalicum
control
strain
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陈德鑫
夏长剑
邓海滨
曹梅
林北森
向小华
吕洪坤
马光祥
王兴高
蔡斌
高华军
耿召良
邵雨
王娟
郭栋
颜统晶
吴少英
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Hainan Co Of China National Tobacco Corp
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Hainan Co Of China National Tobacco Corp
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    • A—HUMAN NECESSITIES
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention discloses a penicillium oxalicum (Penicillium oxalicum) MS01 and application thereof, wherein the strain is preserved in the microorganism strain preservation center of Guangdong province, the preservation date is 2023, 1 month and 4 days, and the preservation number is CDMCC No.63109. The strain has prevention effect on prodenia litura and tobacco budworms, and the prevention effect increases with time in a short period. Compared with the traditional strain which can only control a single prodenia litura, the penicillium oxalicum MS01 can simultaneously control two lepidoptera pests, namely prodenia litura and tobacco budworms, and can effectively save the labor, material resources and time cost required by simultaneously controlling the prodenia litura and the tobacco budworms.

Description

Penicillium oxalate MS01 and application thereof
Technical Field
The invention relates to penicillium oxalicum MS01 and application thereof, and belongs to the technical field of prodenia litura and tobacco budworm control.
Background
For a long time, lepidoptera pests have serious threat to crop yield, quality safety and the like in agricultural production, and the main control means of lepidoptera pests at present mainly comprise chemical control, physical control, agricultural technology and the like, and the methods have certain control effects on lepidoptera pests, but still have the problems of low target pertinence, high control cost, environmental pollution and the like. The biological pest control method has the advantages of small environmental safety risk, lasting control effect, easiness in cooperative application with other plant protection technologies and the like, and the advantages of low pest control cost, high pertinence, convenience, quickness, environmental friendliness and the like by utilizing the entomopathogenic microorganisms are utilized.
The prodenia litura and the tobacco budworms belong to lepidoptera, are agricultural pests with great harm, are world pests distributed in tropical and subtropical areas, can be 100 or more than 289 plants such as harmful fruits and vegetables, weeds, ornamental plants for gardens and the like, and have the characteristics of wide distribution, edibility impurities, strong propagation, rapid outbreak and the like. In most areas of China, prodenia litura can take multiple generations each year, larvae are often gathered together, tender plant tissues are taken, the larvae begin to take the feed in a dispersed manner from 3-year-old larvae, stalks, flower buds and fruits can be eaten, crop harvest is easy to cause, economic loss is caused, and yield reduction can sometimes be caused to exceed 25%. In the long-term chemical control process, serious drug resistance is generated, the sensitivity to various agents is reduced, great difficulty is caused in the control process, the control time can be prolonged, the population outbreak can be restrained, pathogenic bacteria with lethal effect on lepidoptera pests are explored from lepidoptera pest dead bodies, the insecticidal effect is improved, and a new control way and thinking can be provided for the lepidoptera pests.
Patent literature (CN 113150999 a) discloses a fungal strain of insect living with high pathogenicity to prodenia litura and application thereof, the scheme obtains an excellent strain SCAUYZ-16 with high pathogenicity to prodenia litura through screening research, however, the strain only has a pathogenicity to one target organism of prodenia litura, and in actual production, the simultaneous control of the prodenia litura and the prodenia litura cannot be realized, and the simultaneous control of several pests can be realized only by screening and simultaneous application of pathogenic bacteria of the prodenia litura and the prodenia litura again, so that a great amount of manpower, physical and time costs are required.
Disclosure of Invention
In order to solve the problem that the current SCAUYZ-16 strain can only control prodenia litura but can not control tobacco budworms at the same time, the invention provides the penicillium oxalicum MS01, and the penicillium oxalicum MS01 has pathogenicity on both the prodenia litura and the tobacco budworms, and can control both pests at the same time, so that the cost of people, material resources and time is saved.
The inventor obtains a bacterial strain capable of simultaneously preventing and controlling prodenia litura and tobacco budworms from body surface spores of morbid prodenia litura adults. This strain was identified as penicillium oxalicum MS01, which strain constitutes the first aspect of the invention.
The strain is used for controlling prodenia litura and/or tobacco budworms, which form a second aspect of the invention. Wherein it is preferred that the penicillium oxalicum is used in the form of conidia. More preferably, the conidia are present in the form of a suspension or a spore powder. The use concentration of the penicillium oxalicum is 1 multiplied by 10 7 ～1×10 9 spores/mL.
The pesticide containing penicillium oxalicum as an active ingredient constitutes a third aspect of the present invention. Preferably, the penicillium oxalicum is used in the form of conidia. More preferably, the conidia are present in the form of a suspension or a spore powder. The use concentration of the penicillium oxalicum is 1 multiplied by 10 7 ～1×10 9 spores/mL.
The beneficial effects of the invention are as follows: the screened penicillium oxalicum MS01 has high control effect on prodenia litura and tobacco budworms, and the control effect increases with time in a short period. Compared with the traditional strain which can only control a single prodenia litura, the penicillium oxalicum MS01 can simultaneously control two lepidoptera pests of the prodenia litura and the tobacco budworms, the prodenia litura and the tobacco budworms are main pests of tobacco, and the two pests frequently occur and damage in tobacco fields.
The strain of the invention has a preservation date of 2023, 1 month and 4 days and a preservation number of CDMCC No.63109. The classification is named as penicillium oxalicum (Penicillum oxalicum) MS01, the preservation unit name is the collection of microbiological bacterial strains in Guangdong province (CDMCC for short, and the address is building 5, building 59, guangzhou Miao Jiujia 100, and the postal code is 510070.
Drawings
FIG. 1A shows the form of the mycelium of Penicillium oxalicum MS 01;
FIG. 2 shows the result of electrophoresis detection of amplified DNA fragments;
FIG. 3MS01 BLAST comparison results.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1: isolation and purification of Penicillium oxalicum
Placing adult prodenia litura suffering from diseases in a sterilization culture dish, culturing in a constant temperature incubator at 28 ℃, after the conidium grows obviously on the surface of the adult, lightly picking a small amount of hyphae or spores from the surface of the adult in a disinfection and sterilization ultra-clean workbench by using an inoculating loop, inoculating the mycelia or spores on a potato dextrose agar medium PDA (200 g of potato, 20g of dextrose, 15g of agar powder and 1000mL of water) by adopting a shaking method, and culturing in the constant temperature and humidity incubator at 28 ℃ with the relative humidity of 70%. After 5d of culture, the pollution-free mycelium or spore is selected and inoculated to a new culture medium for purification culture until the morphological characteristics of colonies growing on the flat plate are consistent. After the purified and cultivated strain fully produces spores, the strains are stored in a refrigerator at the temperature of 4 ℃ for standby.
Example 2: identification of Penicillium oxalicum
1. Morphological identification
A small amount of mycelia in example 1 was picked up in an ultra clean bench, placed on a slide glass with distilled water dropwise and covered with a cover glass, and the conidium and conidium stalk morphology of the fungus were observed with a Zeiss microscope for a total of 5 fields of view. The mycelium morphology of Penicillium oxalicum MS01 is shown in FIG. 1.
ITS/ITS4 molecular characterization
The bacterial DNA extraction method comprises the following steps:
1) 20mg of mycelia were ground to a powder with liquid nitrogen on a super clean bench and added to a 1.5mL centrifuge tube. 400 mu LBuffer Digestion and 4 mu L of beta-mercaptoethanol are added, and the mixture is mixed by shaking. The cells were completely lysed in a water bath at 65℃for 1 h.
2) 200. Mu.L Buffer PF was added and mixed well by inversion and left in a refrigerator at-20℃for 5min.
3) Centrifuge at 10000rpm for 5min at room temperature, transfer supernatant (500-550. Mu.L) to a new 1.5mL centrifuge tube.
4) Adding equal volume of isopropanol, reversing for 5-8 times to mix thoroughly, and standing at room temperature for 2-3min. Centrifuge at 10000rpm for 5min at room temperature, discard supernatant.
5) 1mL of 75% ethanol was added, rinsing was reversed for 1-3min, centrifugation was performed at 10000rpm for 2min, and the supernatant was discarded.
6) Repeating step 5) once.
7) The cover of the super clean bench is opened and inverted for 5-10min at room temperature until the residual ethanol is completely volatilized.
8) The obtained DNA was dissolved in 50 to 100. Mu. LTE Buffer.
PCR amplification and agarose gel electrophoresis:
the total volume of the PCR reaction system was 25. Mu.L, including 2. Mu.L of DNA solution, 12.5. Mu.L of Mix solution, 1. Mu.L of each of the primers ITS1 and ITS4, and ddH 2 O8.5. Mu.L. Adding samples for amplification according to the program set in Table 1; preparing agarose gel, adding nucleic acid dye, after the agarose gel is solidified, sequentially pumping DL2000Plus DNAMaroer and amplified PCR product into a gel hole by using a pipette, and carrying out electrophoresis under 160V voltage. The result of the electrophoresis detection of the DNA amplified fragments is shown in FIG. 2. The sequencing results were aligned with the BLAST database and the alignment results are shown in figure 3, which shows that it belongs to Penicillum oxalicum.
TABLE 1PCR reaction cycle parameters
Step (a) Temperature (. Degree. C.) Time(s)
Pre-denaturation 94 300
Denaturation (denaturation) 94 30
Annealing 56 30～35cycles
Extension 72 45
Terminating the extension 72 600
MS01 ITSI/ITS4 sequencing sequence:
TATCGGAGTGAGGGGCCTCTGGGTCCAACCTCCCACCCGTGTTTATCGTACCTTGTTGCTTC
GGCGGGCCCGCCTCACGGCCGCCGGGGGGCATCCGCCCCCGGGCCCGCGCCCGCCGAAGA
CACACAAACGAACTCTTGTCTGAAGATTGCAGTCTGAGTACTTGACTAAATCAGTTAAAAC
TTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTA
ATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTAT
TCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGC
TCTCGCCCCCCGCTTCCGGGGGGCGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCC
TCGAGCGTATGGGGCTTCGTCACCCGCTCTGTAGGCCCGGCCGGCGCCCGCCTGCGAACAC
CATCAATCTTAACCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA, SEQ ID NO.1.
The above primers ITS1 and ITS4 are universal primers, and specific nucleotide sequences are as follows: ITS1:5'-TC CGTAGGTGAACCTGCGG-3'; ITS4:5'-TCCTCCGCTTATTGATATGC-3' is shown as SEQ ID NO.2 and SEQ ID NO. 3 respectively.
Based on the sequencing result and the physiological and biochemical test result, the MS01 strain is determined to belong to Penicillum oxalicum. Therefore, the strain was designated as Penicillum oxalicum MS01 and deposited with the Guangdong province microorganism strain collection center (CDMCC: no. 59 building 5 of the university of Mitsui, guangdong province, university of science, microorganism institute, postal code: 510070) at 2023, 1 month and 4 days with deposit number CDMCC No.63109.
Example 3: penicillium oxalate MS01 insecticidal effect
Example 1 hyphae and spore powder were scraped from a petri dish incubated for 2 weeks with a sterile inoculating loop, scraped and placed in a sterile 15mL centrifuge tube and added with 5mL of 0.05% tween-80 and shaken well for 30min. Dilution of fungal spore suspension to 1X 10 with 0.05% Tween-80 by dipping 8 The spores/mL are picked up by using sterilizing forceps, the spodoptera litura and the 2-year larva of the tobacco budworms are soaked in culture dishes (the diameter is 9cm and the height is 2 cm) to prepare suspension liquid with various concentrations for 2s, taken out, put on filter paper, crawled and air-dried, and then put in a feeding box. Each treatment of 20 head 2 instar larvae was repeated 3 times with 0.05% tween-80 as a control. The feeding box is placed in a artificial climate box (T=27+ -1 ℃, RH=75++5%, L: D=14)h: 10 h) and feed was changed every 24h and observed for 7 days, and the number of deaths was recorded. The larvae were gently touched with a small brush pen and the larvae were rendered weak and unable to crawl normally as a record of death. The results are shown in Table 2.
TABLE 2 killing of lepidopteran pests by Penicillium oxalate MS01 spore suspension
Test insects 1d 2d 3d 4d 5d 6d 7d
Prodenia litura (L.) DC 7.1±2％ 10.3±1％ 20.6±2％ 35.4±3％ 70.2±2％ 78.3±3％ 86.4±3％
Tobacco budworm 5.6±1％ 15.5±2％ 20.4±1％ 30.3±2％ 55.9±3％ 75.4±3％ 83.3±2％
Table 1 shows that the penicillium oxalicum MS01 has better insecticidal toxicity to the prodenia litura and the tobacco budworms, the 1 st day after treatment starts to play a lethal effect, the 3 rd to 5 th days are the infection peak period of pathogenic bacteria, the mortality rate is obviously improved, and the mortality rates of the 7d, MS01 bacterial spore suspension to the prodenia litura and the tobacco budworms are 86.4% and 83.3% respectively.
Example 4: penicillium oxalicum MS01 field control effect
A separate experiment was performed in the university of hainan, meadow school district, 2021.
Test time: 2021.
Test site: cigar field at farm base of university of Hainan, U.S. Kakou, min's way, 58.
Test material: penicillium MS01
Test object: prodenia litura and tobacco budworms
The test was carried out in cigar fields. Let 1X 10 8 spore/mL penicillium MS01 spore suspension, with equal amount of CK (clear water) as control, each treatment was repeated 3 times, each cell area was 28m 2 (4 m is multiplied by 7 m), 10L of liquid medicine is sprayed in each cell, and 1 ridge is reserved between the cells and is not subjected to any treatment, so that the liquid medicine is used as a buffer protection area. All cells were uniformly sprayed using a hand-held knapsack agricultural sprayer. No other insecticide was used during the test, and the field management of each plot was consistent with other vegetable plots.
The number of prodenia litura and tobacco budworms in each cell was investigated by a 5-point sampling method, and 5 plants were investigated per point. 1 time was investigated on the day before administration, and 1 time was investigated on the 3 rd, 5 th, 7 th and 14 th days after administration. The numbers of prodenia litura and tobacco budworms of each plant are recorded respectively. And calculating the insect population density (number of insects/hundred plants), the insect population decline rate and the relative prevention effect according to the statistical result.
The rate of reduction of insect population (%) = (number of live insects before drug-number of live insects after drug)/(number of live insects before drug) ×100;
control effect (%) = (treatment area reduction rate-blank treatment reduction rate)/(100-blank treatment reduction rate) ×100
TABLE 3 field control of Penicillium MS01 spore suspension on Spodoptera litura
As shown in table 3, the investigation results of 3, 5, 7 and 14d show that the withdrawal rates of the control groups are-14.3%, -26.2%, -30.1%, -36.7, respectively, and the control groups have no control effect on prodenia litura; the degradation rates of the penicillium oxalate on the 3 rd, 5 th, 7 th and 14 th d prodenia litura are sequentially increased and are respectively 23.4%, 57.4%, 82.9% and 89.8%, and the relative prevention effects are respectively 33.0%, 66.2%, 86.8% and 92.5%. Therefore, the penicillium oxalicum has a prevention effect on prodenia litura.
TABLE 4 field control of Penicillium MS01 spore suspension on Firework
As can be seen from table 4, the investigation results of 3, 5, 7 and 14d show that the degradation rates of the control groups are respectively-9.2%, -20.2%, -24.9%, -30.1%, and the control groups have no control effect on the tobacco budworms; the degradation rates of the penicillium oxalate on the 3 rd, 5 th, 7 th and 14 th days of the penicillium fumigatus are sequentially increased, and are respectively 15.3%, 46.8%, 69.9% and 81.8%, and the relative prevention effects are respectively 22.4%, 55.7%, 69.9% and 86.0%. Therefore, the penicillium oxalicum has a prevention effect on the tobacco budworms.
In conclusion, when the penicillium oxalicum disclosed by the invention is applied to a tobacco field, the 5 th and 7 th effects of the penicillium oxalicum on the spodoptera litura are 66.2% and 86.8% respectively, and the 14 th effect of the penicillium oxalicum is 92.5%, so that the penicillium oxalicum can exert a better control effect on the spodoptera litura in the field; the control effect on the tobacco budworms is obviously improved from 3 rd to 5 th days, and the 14 th control effect reaches 86.0%, which shows that the bacterium can exert better control effect on the tobacco budworms in fields and has development and application potential.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

1. Penicillium oxalicum (Penicillium oxalicum) MS01 deposited at the Cantonese collection of microorganism strains at 2023, 1 and 4 days and with a deposit number of CDMCCNo.63109.
2. The use of the penicillium oxalicum MS01 according to claim 1 for controlling prodenia litura and/or tobacco budworms.
3. The use according to claim 2, characterized in that the penicillium oxalicum MS01 is used in the form of conidia.
4. Use according to claim 3, wherein the conidia are present in the form of a suspension or a spore powder.
5. The use according to claim 3, wherein the Penicillium oxalate MS01 is used at a concentration of 1X 10 7 ～1×10 9 spores/mL.
6. A pesticide containing penicillium oxalate MS01 as an active ingredient is characterized in that the penicillium oxalate MS01 is preserved in the microorganism strain collection of Guangdong province, the preservation date is 2023, 1 month and 4 days, and the preservation number is CDMCCNo.63109.
7. The pesticide according to claim 6, wherein the penicillium oxalicum MS01 is used in the form of conidia.
8. The pesticide according to claim 6, wherein the conidia are present in the form of a suspension or a spore powder.
9. The pesticide according to claim 6, wherein the Penicillium oxalate MS01 is used at a concentration of 1X 10 7 ～1×10 9 spores/mL.
CN202310450434.0A 2023-04-25 2023-04-25 Penicillium oxalate MS01 and application thereof Pending CN116606742A (en)

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