CN110894469B - Metarhizium anisopliae strain with high pathogenicity on orange belt mythimna separata larvae and application thereof - Google Patents
Metarhizium anisopliae strain with high pathogenicity on orange belt mythimna separata larvae and application thereof Download PDFInfo
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Abstract
The invention discloses a metarhizium anisopliae strain with high pathogenicity on orange belt mythimna separata larvae and application thereof, belonging to the technical field of biological control. The strain is named as Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa201516, with the preservation number of CGMCC No. 18123. The strain has high growth speed and strong spore production capability, and has high pathogenicity on orange band gyplusia melanoides larvae, which is 10 percent7After the spore suspension of spores/mL is inoculated on larvae of the orange band Plutella xylostella for 15 days, the cumulative death rate of the larvae reaches 96.7 percent, and LT is50For 5.01 days; the fungus preparation produced by the strain has good control effect on the orange band gyplutella melanosticta and can effectively reduce the population density of the larvae of the orange band gyplutella melanosticta. The strain has potential value in the prevention and treatment of the orange band gyplusia operculella, and has positive significance in reducing the harm caused by the orange band gyplusia operculella and reducing the using amount of chemical pesticides.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a metarhizium anisopliae strain with high pathogenicity on orange band mythimna separata larvae and application thereof.
Background
Arhat pine (A) and (B)Podocarpus macrophyllus) And bamboo bark (bamboo bark, cypress), (bamboo bark, cypress, bamboo bark, bambooPodocarpus nagi ) Is an important tree species for garden greening and garden landscape in ChinaEspecially, the podocarpus macrophyllus has high economic value, and China needs to introduce a great amount of species from abroad every year. Orange with Langeye (Bombycis Mori)Milionia basalisWalker) genus Lepidoptera (Lepidotera) family Geometriaceae (Geometridae) genus Neurospora (Geometridae)Milionia Walker 1854) is an important leaf-eating pest which occurs in southern China in recent years on Japanese pines and bamboo cypresses, leaves of host plants are used up when serious occurrence occurs, only bald branches are left, and occurrence and serious harm reports are generated in China, Guangxi, Guangdong, Hainan, Taiwan, Fujian and other areas and Japan abroad. At present, no effective means exists for preventing and controlling the insects, and the method is mainly used for spraying chemical pesticides during outbreak. Therefore, it is necessary to develop a control technology, especially a biological control technology, for the insect.
Metarrhizium anisopliae is an entomogenous fungus which is widely applied, has a wide host spectrum, and can parasitize more than 200 insects, mites, nematodes and the like. The metarhizium anisopliae has the advantages of strong adaptability, long-term storage in soil and forest, easy large-scale production, no toxicity to people and livestock, environmental friendliness and the like, and plays an important role in the biological control of agricultural and forestry pests. The strain with high pathogenicity to the target pests is the basis for the application of the fungal pesticide, and the excellent strain not only has good growth characteristics and can be produced in large scale, but also has strong pathogenicity to the target pests. Through retrieval, no report on the control of the orange belt black-foot moth by using metarhizium anisopliae exists at present. Based on the above, in order to provide biological control resources of the orange band mythimna separata, the invention screens out excellent metarhizium anisopliae strains with strong pathogenicity to the larvae of the orange band mythimna separata, provides excellent strains and microbial inoculum for production, and has positive significance for efficiently controlling the orange band mythimna separata, reducing the harm caused by the orange band mythimna separata and reducing the usage amount of chemical pesticides.
Disclosure of Invention
The invention aims to provide a metarhizium anisopliae strain with high pathogenicity on orange belt mythimna separata larvae and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a strain of Metarhizium anisopliae, its classification name is Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa201516 strain, deposited at China General Microbiological Culture Collection Center (CGMCC) on 26.8.2019, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation number is: CGMCC No. 18123.
The Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa2011516 strain, having the following characteristics:
the strain grows for 5 days on a PDA culture medium at 25 +/-1 ℃, the diameter of a colony is 23mm, and the diameter of the colony reaches 62mm in 15 days. The colony is white in the initial stage and is villous to flocculent in texture; gradually enlarge, produce dark green conidium on it and distribute in a ring shape, and are powdery. Hyphae are branched and have lattices, are colorless and smooth, have the width of 2.3 to 3.2 mu m, conidiophores are single or branched, and the tops of the branches have 2 to 5 columnar spore-forming cells. Conidiophore single cell is oblong and smooth in surface, and is aggregated into spore mass with size of (4.5-6.8 μm) × (1.8-2.9 μm). The optimal growth temperature range is 25-30 ℃.
The invention aims to solve another technical problem of providing a bacterial strain and a microbial inoculum for controlling larvae of the orange band Plutella xylostella.
The microbial inoculum provided by the invention is conidium suspension. Wherein the spore suspension has a concentration of 107Spores/ml.
The effective component of the microbial inoculum provided by the invention is the metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Conidia of FJMa 201516.
The preparation method of the conidium suspension in the microbial inoculum provided by the invention comprises the following steps: metarhizium anisopliae (Metarhizium anisopliae, MetarhiziumMetarhizium anisopliae) The FJMa201516 strain is inoculated into an SDY liquid culture medium under the aseptic condition and is subjected to shake culture at 25 +/-1 ℃ for 72 h, then is inoculated into a PPDA plate for culture for 9-10 days, and is stored for later use after sufficient spore production. Scraping a certain amount of spores from the plate, placing in sterilized 0.3 ‰ Tween-80 solution, and making into 1 × 107spores/mL of spore suspension.
The microbial inoculum provided by the invention can be used for preventing and controlling larvae of the orange band gyplusia ni.
The metarhizium anisopliae (Metarhizium anisopliae) provided by the inventionMetarhizium anisopliae) The FJMa201516 strain has the following beneficial effects:
the strain has good production property, strong spore production capability and strong pathogenicity to orange belt gypsy moth larvae, and the adopted concentration is 107After 15 days of inoculation with spore suspension of spores/ml, the mortality rate of larvae of the orange band Plutella xylostella is 96.7%, and the larvae are Lethal (LT)50) It was 5.01 days. The microbial inoculum produced by the strain can obviously reduce the population quantity of orange band gyplusia larvae in forests. Meanwhile, the strain can survive in soil for a long time, and has a remarkable control effect on pupae of the orange band gypsy moth pupae which pupates in the soil. The microbial inoculum of the strain is environment-friendly and efficient, has a continuous control effect, can greatly reduce the use amount of chemical pesticides, and has a good application prospect.
The strain name is as follows: metarhizium anisopliae
Latin learning name:Metarhizium anisopliae
the strain number is as follows: FJMa201516
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 26/8/2019
Registration number of the preservation center: CGMCC No.18123
Description of the drawings:
FIG. 1 is a colony morphology of Metarhizium anisopliae FJMa201516 strain cultured on PPDA medium at 25 + -1 deg.C for 15 days.
FIG. 2 shows healthy orange-banded mythimna larvae and their symptoms infected with Metarhizium anisopliae FJMa 201516. a, healthy larvae of orange with gypsy moth; b, infection by Metarhizium anisopliae FJMa 201516.
Detailed Description
The following detailed description of specific embodiments of the present invention is provided in conjunction with specific examples which are intended to be illustrative, but not limiting, of the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The PPDA medium in the following examples is a peptone-potato-glucose agar medium, abbreviated as "English", and its formulation is such that it contains 200g of potato (cut pieces are boiled and filtered, and the filtrate is taken out), 20g of glucose, 10g of peptone, and 20g of agar per 1000ml of water.
Example 1 Metarhizium anisopliae (Metarhizium anisopliae) (II)Metarhizium anisopliae) Identification of FJMa201516
Morphological identification:
culturing the separated and purified metarhizium anisopliae FJMa201516 strain on a flat PPDA culture medium with the diameter of 90mm at the temperature of 25 +/-1 ℃, picking white hyphae after 4-5 days, and flaking the hyphae and conidiophore forms of the strain under a microscope; and (4) picking mature conidia after 8-10 days, and slicing the conidia and observing the shape and size of the conidia under a microscope. The colony morphology characteristics are observed and recorded for about 15 days. The bacterial colony of the strain is white in initial stage and cotton-like in texture, and dark green conidia are generated on the bacterial colony after 4-5 days and are distributed in a ring shape. Hyphae are branched and have lattices, are colorless and smooth, have the width of 2.2 to 3.3 mu m, have single or branched conidium peduncles, have 2 to 5 columnar spore-producing cells at the top of the branches, and generate inverted conidium chains on the conidium peduncles. Conidium single cells are oblong, have smooth surfaces and have the size of 4.4-6.7 mu m multiplied by 1.8-3.0 mu m.
ITS sequence amplification, sequence determination and molecular identification of strain
Using Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Extracting total DNA from mycelia of FJMa201516 strain, and performing PCR amplification by using the extracted DNA as a template and universal primers ITS4 and ITS5 (synthesized by Shanghai Biotech, Ltd.) in an ITS zone, wherein 2 XTaq PCR MasterMix (containing dye) is 12.5 muL; 1 muL each of the primers ITS4/ITS5 (10 mumol/L); ddH2O9.5 muL; template DNA 1 μ L. PCR reaction procedure: pre-denaturation at 95 ℃ for 4min, denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, and extension at 72 ℃ for 40s, for 35 cyclesFinally, extension was carried out at 72 ℃ for 7min, and sterile water was used instead of the template as a negative control. And after the PCR product is detected by an electrophoresis strip, confirming that the PCR amplification product of the DNA-ITS region of the sample meets the sequencing requirement, connecting and converting the PCR amplification product, and then sending the PCR amplification product to a platane biotechnology (Shanghai) company Limited for sequence determination. The sequencing results were compared to metarhizium strain sequences in the National Center for Biotechnology Information (NCBI) Gene database.
Amplification primer sequences:
ITS4:5'-TCCT CCGC TTAT TGAT ATGC-3' 。
ITS5：5'-GGAA GTAA AAGT CGTA ACAAGG-3' 。
the ITS sequence of the metarhizium anisopliae strain is shown as SEQ ID NO.1, and the sequence length is 555 bp. Discovered to be related to Metarhizium anisopliae after NCBI/blast comparison (Metarhizium anisopliae) MH855048.1 and FJ545320.1 have the highest homology of 100 percent and 99.82 percent respectively, and the strain is confirmed to be Metarhizium anisopliae (Metarhizium anisopliae) ((R))Metarhizium anisopliae）。
Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) The FJMa201516 strain is preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 8 and 26 months, and the preservation number is CGMCC No. 18123.
Example 2 Metarhizium anisopliae (Metarhizium anisopliae) (II)Metarhizium anisopliae) Biological Properties of FJMa201516
Metarhizium anisopliae (Metarhizium anisopliae, MetarhiziumMetarhizium anisopliae) FJMa201516 was spotted onto the center of PPDA plate medium, cultured in an incubator at 25 + -1 deg.C, and the colony diameter was measured 1 time per day by the cross method, 3 plates (replicates) were set for each treatment, and observed for 15 days. At the same time, the time of starting spore production is recorded, a small bacterial dish is punched from the center of a bacterial colony to the edge 1/2 by a puncher with the diameter of 1.5cm on the 15 th day (the spore production is basically finished), the bacterial dish is taken out and placed in a triangular flask containing 30 mL of 0.3 per thousand Tween-80 sterile water, and the flask is oscillatedSpores were sufficiently dispersed and appropriately diluted, and the spore concentration was measured with a hemocytometer and converted into the amount of spores produced per unit area. The assay was repeated 3 times per plate, and the average value was taken as the number of spores in 1 replicate.
TABLE 1 Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa201516 biological Properties
As shown in Table 1, Metarhizium anisopliae (Metarhizium anisopliae) ((Metarhizium anisopliae) FJMa201516 colony grows fast, the diameter of the colony is 23mm in 5 days, the diameter of the colony reaches 62mm in 15 days, green spores begin to appear on the colony after the colony grows for 3 days, and the spore yield is 1.33 multiplied by 108Spore/cm2。
Example 3
Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Determination of pathogenicity of FJMa201516 to Oreomys nivea larvae
Collecting and feeding test insects:
the test insects are larvae of the orange tape gypsy moth, and are collected in a nursery of the Japanese cypress in Youyi county 5, 12 months in 2019, and the Japanese cypress is seriously damaged by the orange tape gypsy moth in the nursery. The collected larvae are taken back to a laboratory, placed in an insect cage, and fed with fresh podocarpus macrophyllus leaves as feed. After 1 day, selecting larvae with normal activities and 3-4 instars, placing the larvae in an insect rearing box, and placing 15 larvae in each rearing box for measuring the pathogenicity of the metarhizium anisopliae.
Determination and analysis of pathogenicity
Scraping a certain amount of Metarhizium anisopliae from PPDA (PPDA) plateMetarhizium anisopliae) FJMa201516 spore is put into sterilized 0.3 per mill tween-80 solution to be prepared into 107spores/mL of spore suspension; inoculating by spraying method, namely spraying the prepared spore suspension on the surface of larva body uniformly, and spraying with sterile water as contrast. 15 larvae are inoculated in 1 repeat, the inoculation amount of each repeat is 1ml of spore suspension, 4 repeats are set, and 60 test insects are inoculated in total. After the treatment is finished and the worm bodies are slightly dry, feeding the fresh podocarpus macrophyllus branches and leaves. After inoculation every timeObserving the death condition of the test insects every day, replacing the feeding branches and leaves every 2 days, taking the condition that no reaction exists when the test insects are touched as the death standard, timely moving the dead test insects out of the insect box, placing the insect box in a sterilized culture dish for moisture preservation culture, observing whether the insects grow hypha and produce spores, and determining whether the insects are infected by the metarhizium anisopliae.
Meanwhile, the pathogenicity of different strains to the larvae of the orange belt blue foot moth is compared by taking inoculated metarhizium anisopliae FJMa201101 (invention patent: 201510055121.0) with high pathogenicity to the larvae of the Camellia sinensis elephant and a metarhizium anisopliae strain FJMa200902 (invention patent: 201510559073.9) with pathogenicity to Laos quasipaa spinosa cattle as a comparison.
Statistical analysis of data was performed using the DPS data processing system software to calculate larval mortality and infection rates 15 days after inoculation based on observations. Adopting DPS self-carried mortality-time probability value analysis module to calculate the pathogenicity of metarhizium anisopliae to orange band Langmuia larvae, namely taking the logarithm value of time (d) as x, converting the death rate into probability value, taking the probability value as y, simulating a pathogenicity regression equation, and calculating the death time (LT) by taking the probability value as y50）。
By using 107And (3) inoculating the larvae of the orange band blue-foot moth inoculated with FJMa201516 strain after 15 days of spore suspension of the spores/mL, wherein the mortality rate of the larvae of the orange band blue-foot moth inoculated with FJMa201516 strain reaches 96.7%, and the death peak is 3-7 days. The 15-day mortality rates of orange-banded mythimna larvae inoculated with metarhizium anisopliae and FJMa201101 and FJMa200902 strains were 83.3% and 85%, respectively, lower than those inoculated with FJMa201516 strain. The mortality rate after 15 days in the blank control group was 10%. Most of the larvae of the experimental group are infected and killed by the metarhizium anisopliae, and the blank control group has no infection. After the spore suspension is inoculated by dipping method, metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa201516 was 5.01 days lethal to orange band diptera larvae. The pathogenicity of the metarhizium anisopliae FJMa201101 and FJMa200902 strains to the orange belt blue foot moth larvae is 7.83 days and 7.12 days respectively, and is obviously longer than the middle lethal time of inoculation of FJMa 201516. The results of the inoculation test also show that the pathogenicity of the metarhizium anisopliae FJMa201516 to the larvae of the orange band blue foot moth is higher than that of the metarhizium anisopliae FJMa201101 (invention patent: 201510055121.0) and that of the metarhizium anisopliae FJMa201516Metarhizium anisopliae strain FJMa200902 (invention patent: 201510559073.9) is strong.
TABLE 2 cumulative mortality of orange-banded Dipper larvae inoculated with different metarhizium anisopliae strain spore suspensions
TABLE 3 pathogenicity analysis of different Metarhizium anisopliae strains on orange band Langerhans larva
Example 4
Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) Forest control effect of FJMa201516 spore suspension on orange band gyplusia larvae
Metarhizium anisopliae (Metarhizium anisopliae) obtained by solid fermentation on solid culture medium containing testa Tritici, rice and testa oryzae (at volume ratio of 1: 1: 1) as main ingredientsMetarhizium anisopliae) FJMa201516 spores are prepared into 10 by a dilution method7spore/mL spore suspension, and the preparation solvent is 0.3 per mill of Tween-80 solution, and the microbial inoculum is prepared for use on site to ensure the effect. After the preparation, the conventional motor-driven sprayer is directly used for spraying in the forest, the leaf surface and the leaf back of the larvae of the gypsy moth with orange bands are aimed at to spray in all directions by taking the spraying of the leaf surface as the standard, and the spraying is carried out when the temperature in the shade is not more than 30 ℃. After spraying, selecting the larvae of the orange band gypsy moth in the forest for cage observation, and ensuring that no less than 30 larvae of the orange band gypsy moth are sleeved in each cage for 5 cages. Larval mortality was observed at 5 days and 15 days, respectively.
The forest control effect (Table 4) shows that the spore suspension (with the concentration of 10) is prepared by the metarhizium anisopliae FJMa2015167spore/mL) is sprayed, the population density of the forest orange band gypsy moth larvae is greatly reduced along with time, wherein the population reduction rate in 5 days is 25.61%, the population reduction rate in 15 balances reaches 91.60%, and the forest control effect is good. In the forestWhen the prevention and control effect is observed, the activity of the larvae of the orange with the gypsy moth is obviously reduced after 5 days, the food intake is obviously reduced, although the prevention and control effect is counted by the death of pests, the prevention and control effect is actually better than the observed value.
TABLE 4 Metarhizium anisopliae FJMa201516 spore suspension spray control effect on orange band Langerhans larvae
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
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gccgatcccc aacaccaagt cccacagggg acttgagggg cgtaatgacg ctcgaacagg 240
catgcccgcc agaatactga cgggcgcaat gtgcgttcaa agattcgatg attcactgaa 300
ttctgcaatt cacattactt atcgcatttc gctgcgttct tcatcgatgc cagaaccaag 360
agatccgttg ttgaaagttt tgattcattt tttttaacca ctcagaagat acttattaaa 420
aaattcagaa ggtttgggtc cccggcgggc gcgaagtccc gccgaagcaa caattaaagg 480
tataattcac aggggttggg agttggataa ctcggtaatg atccctccgc tggttcacca 540
acggagacct tgtta 555
Claims (5)
1. A metarhizium anisopliae strain with high pathogenicity to orange belt mythimna separata larvae is characterized in that: the strain is as follows: metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) FJMa201516, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 8 and 26 months, with the preservation number of CGMCC No. 18123.
2. A fungal pesticide, which is characterized in that: the fungal pesticide comprises the metarhizium anisopliae (Metarhizium anisopliae) (of claim 1)Metarhizium anisopliae) Strain FJMa 201516.
3. The fungal pesticide of claim 2, wherein: the dosage form is conidium suspension.
4. The fungal pesticide of claim 2, wherein: the effective component is conidium of Metarhizium anisopliae with concentration of 1 × 107spores/mL.
5. The use of a metarhizium anisopliae strain of claim 1 having high virulence for larvae of the orange-banded mythimna livida for controlling larvae of the orange-banded mythimna livida.
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