CN106479943A - The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application - Google Patents

Abstract

The invention discloses a strain of Isaria javanica for controlling white moth, which is classified as Isaria javanica ( Isaria javanica ) BE01, and is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC No.13192 , the preservation date is November 28, 2016, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The I. javanica BE01 strain of the present invention is isolated from the larvae of the white moth larvae, and it is confirmed by experiments that it has a good effect on the spore concentration of 1.0× ¹⁰⁴ /mL or more for the white moth of different ages. The virulence, the infection rate exceeds 53.33%, and at the spore concentration of 1.0×10 ⁸ /mL, the infection rate exceeds 73.33%, which can effectively control the white moth.

Description

A strain of I. javanica BE01 highly effective in controlling white moth and its application

technical field

The invention belongs to the technical field of microbes, and in particular relates to strains of I. javanica and its application in pest control.

Background technique

American white moth (Hyphandria cunea), also known as autumn curtain caterpillar and autumn curtain moth, belongs to Lepidor tera, Arctiidae and Arctiinae, and is an important species in many countries. pests. The worm is native to North America, and spread to Europe and Asia through human activities and delivery vehicles in the late 1940s. The American white moth was first discovered in Dandong, Liaoning Province, my country in 1979, causing serious damage, and then spread to Beijing, Tianjin, Hebei, Shandong, Shanxi and other provinces and cities. At present, due to the damage of this invasive lepidopteran pest, huge losses have been caused in forestry and fruit trees and even in agriculture. The hosts of this pest include almost all forest trees, fruit trees, ornamental plants, ornamental flowers, vegetables, crops, herbs and shrubs. At the same time, the American Bai'e is small in size but eats a lot, and it continues to eat and cause damage, so it is also called "forest fire without smoke".

In recent years, the control method of American white moth is mainly chemical control. Chemical control is mainly applied to the larvae of American white moth, and it has a good effect on the larvae before the 4th instar, but the effect on the American white moth that breaks through the net and spreads is weakened. And long-term use of chemical pesticides will bring serious environmental pollution and even affect human health.

Chouioia cunea Yang is the main insect enemy of the American white moth, and only Beauveria bassiana and Isaria farinose have been reported to infect the American white moth. But there is no report on using Isaria javanica to control American white moth.

Contents of the invention

Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the invention is to provide a high-efficiency strain of I. japonicus to meet the needs of use. Another object of the present invention is to provide the application of the high-efficiency I. javanica strain to control white moth.

Technical solution: In order to realize the above-mentioned purpose of the invention, the technical solution adopted in the present invention is:

A strain of Isaria javanica for controlling white moth, named Isaria javanica, was preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms, with the preservation number CGMCC No.13192, and the preservation date was November 2016 On April 28, the preservation address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

The application of the described I. javanica BE01 in the control of the white moth.

In the application, the spore liquid of I. javanica BE01 is directly sprayed on the larvae of the white moth.

The preparation method of the spore liquid is as follows: from the isolated and purified I. javanica BE01 strain, pick mycelium and inoculate it on a SDAY plate covered with cellophane, culture it at 25°C for 20 days, and scrape the mycelium on the cellophane and spores were put into 0.1% Tween-80 aqueous solution, fully stirred and oscillated, filtered to obtain spore mother liquor, and the spore concentration was adjusted to 1.0×10 ⁴ /mL～1.0×10 ⁸ /mL. Preferably, the concentration of the spore liquid is 1.0×10 ⁶ spores/mL to 1.0×10 ⁸ spores/mL.

The I. javanica BE01 was cultured in Chapei's medium for 13 days at 25°C.

The present invention adopts the natural screening method to obtain a strain of Isaria javanica BE01 highly effective against the American white moth through the steps of collecting naturally susceptible insects, separating and purifying strains, identifying fungi, and measuring pathogenicity. Morphological characteristics of I. javanica BE01: As shown in Figure 1-Figure 3, the colonies are irregular in shape, white hyphae, purple-gray spore layer, pale yellow on the back, and spores can be seen on extended culture Stem bundle. Under the microscope, the hyphae are transparent, colorless, 0.3-1.2 μm in width, transparent and smooth. The whorls formed by 2-4 phialides grow on the conidiophores. The base of the phialides is elliptical and enlarged, and gradually becomes thinner upwards; the conidia are transparent and smooth, oblong, with pointed ends. Afterwards, molecular identification was carried out, and a phylogenetic tree was constructed, and the strain was finally determined to be Isaria javanica (Isaria javanica) BE01.

Beneficial effects: Compared with the prior art, the I. javanica BE01 strain of the present invention is isolated from the larvae of the white moth larvae, and it is confirmed by experiments that at a concentration of spores above 1.0× ¹⁰⁴ /mL, it is effective against different The age of the white moth has good virulence, the infection rate exceeds 53.33%, and the infection rate exceeds 73.33% under the spore concentration of 1.0×10 ⁸ spores/mL, which can effectively control the white moth.

Description of drawings

Fig. 1 is the morphological diagram of BE01 bacterial strain growing on Chapei's medium for 13 days;

Figure 2 is a diagram of the sporulation structure of the BE01 strain;

Figure 3 is a diagram of the conidia morphology of the BE01 strain.

detailed description

The present invention will be further described in detail below in conjunction with specific examples. The following examples are preferred embodiments of the present invention. The present invention mainly sets forth the inventive concept of the described bacterial strains, and the embodiments are not described one by one in examples, but the present invention is not limited thereto, and any other deviates from the spirit of the present invention Changes, modifications, substitutions, combinations, and simplifications made under the essence and principles should be regarded as equivalent replacements and included in the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial sources unless otherwise specified:

Insects to be tested: American white moth (Hyphandria cunea) larvae: collected in Suzhou City, Anhui Province; American white moth (Hyphandria cunea) larvae: collected in Huai'an City, Jiangsu Province.

The formula of SDAY medium is as follows: 1% yeast extract powder, 4% glucose, 1% tryptone, 2% agar, add sterile water to 1L, and sterilize at 121°C for 20 minutes.

Cha's medium: 0.2% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate heptahydrate, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, plus sterile Quantify the water to 1L, and sterilize at 121°C for 20 minutes.

Example 1 Isolation and identification of I. javanica BE01

I. javanica BE01 was isolated from the larvae of the American white moth collected in Suzhou, Anhui. The method is as follows: the mycelium is picked from the body of the zombie larvae, inoculated on SD AY medium added with ampicillin and streptomycin sulfate, and cultured at 25° C. for 5 days. Pick the hyphae in the center of the formed colony, inoculate them on the SDAY medium added with ampicillin and streptomycin sulfate for purification and culture, and repeat the purification culture for 3 times. One fungus obtained by screening was numbered as BE01.

1) Morphological identification: As shown in Figure 1-3, the purified strain was inoculated on the Chapei medium, the colony was irregular, the hyphae were white, the color of the spore layer was purple-gray, and the back was light yellow, and spores could be seen after prolonged culture. Stem bundle. Under Zeiss microscope observation, the hyphae are transparent, colorless, 0.3-1.2 μm in width, transparent and smooth. The whorls formed by 2-4 phialides grow on the conidiophores. The base of the phialides is elliptical and enlarged, and gradually becomes thinner upwards; the conidia are transparent and smooth, oblong, with pointed ends.

2) Molecular biological identification

The total DNA of I. javanica BE01 was extracted using the CTAB method to obtain the extracted DNA sample; the total DNA of the extracted strain was used as a template for gene amplification, and the fungal EF1-α primer 983F (5'-GCYCCYGGHCAYCGTGAYTTYAT-3') and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3'), PCR amplification, PCR product recovery and purification, ligation transformation and DNA sequencing, the sequence is shown in SEQ ID NO.1. Compared with the EF1-α sequences of all eukaryotes that have been determined in GenBank, the BE01 strain phylogenetic tree was constructed, and the results showed that the BE01 strain was clustered with KT225600. The characteristics of Isaria javanica, so the BE01 strain can be identified as Isaria javanica, named as Isaria javanica BE01.

Example 2 I. javanica BE01 controls American white moth

Preparation of spore suspension: pick the mycelia from the isolated and purified I. javanica BE01 strain and inoculate it on a SDAY plate covered with cellophane, culture it at 25°C for 20 days, scrape the mycelia and spores on the cellophane into 0.1% Tween-80 aqueous solution, fully stirred and oscillated, and filtered to obtain spore mother liquor. Measure the spore concentration with a hemocytometer and adjust the spore concentration to 1.0×10 ⁴ /mL, 1.0×10 ⁵ /mL, 1.0×10 ⁶ /mL, 1.0×10 ⁷ /mL, 1.0×10 ⁸ /mL.

Adopt spraying method, spray the prepared spore liquid of each concentration on the body of 3-5 instar larvae, control spray 0.1% Tween-80 solution, inoculate 10 heads at each concentration of each larvae age, and each process repeats 3 times , the inoculated larvae were placed in a 25°C incubator at 12:12 light and dark, and raised, and the specific death conditions of each treatment were regularly observed and recorded every day, and the dead larvae were placed in a petri dish with sterilized wet filter paper for 25 ℃ with moisturizing cotton balls for moisture cultivation, record the number of dead worms, and the calculation results are shown in Table 1.

Infection rate formula: Infection rate (%) = zombie number/number of tested insects

Table 1 Infection ability of I. javanica BE01 to the 3rd, 4th, and 5th instar larvae of the American white moth

The results showed that I. javanica BE01 was pathogenic to American white moth, and the mortality rate exceeded 53.33% at the concentration of 1.0×10 ⁴ spores/mL. The strain has a good effect on the 3rd and 4th instar larvae of the American white moth.

SEQUENCE LISTING

<110> Nanjing Forestry University Anhui Academy of Forestry Sciences

<120> A strain of I. javanica BE01 highly effective against white moth and its application

<130> 100

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 643

<212> DNA

<213> Isaria javanica

<400> 1

ggagaccaag gccggcaagt ccaccggcaa gaccctcctc gaggctattg acgccatcga 60

gccccccaag cgtccccctcg acaagcccct ccgtcttcct cttcaggatg tctacaagat 120

cggtggtatc ggaacggtgc ccgtcggtcg tgtcgagact ggtatcatca agcccggcat 180

ggtcgtcacc tttgctcctt ccaacgtcac cactgaagtc aagtccgtcg agatgcacca 240

cgagcagctg cccgagggtg ttcccggtga caacgtcggc ttcaacgtga agaacgtttc 300

cgtcaaggaa atccgtcgtg gtaacgtcgc tggtgactcc aagaacgacc ctcccaacgg 360

tgctgcctcc ttcaacgccc aggtcattgt catcaaccac cctggccaga tcggtaacgg 420

ttacgcccct gttcttgact gccaacactgc ccacattgct tgcaagttct ccgagctcct 480

cgagaagatc gaccgccgta ctggtaagtc ggttgagaac aaccccaagt tcatcaagtc 540

tggtgactcc gccatcgtca agatggttct ctccaagccc atgtgcgttg aggctttcac 600

cgactaccccc cctctgggcc gtttcgccgt ccgtgacatg cgt 643

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<213> Artificial

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<223> Primer 983F sequence

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gcyccygghc aycgtgaytt yat 23

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<211> 23

<212> DNA

<213> Artificial

<220>

<223> Primer 2218R sequence

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atgacaccra crgcracrgt ytg 23

Claims (7)

1. A strain of Isaria javanica for controlling white moth, named Isaria javanica ( Isaria javanica ) BE01, was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms, with the preservation number CGMCCNo.13192 and the preservation date On November 28, 2016, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. 2. The application of I. javanica BE01 described in claim 1 in the control of white moth. 3. The application according to claim 2, characterized in that: the spore liquid of I. javanica BE01 is directly sprayed on the larvae of white moth. 4. The application according to claim 3, characterized in that: the preparation method of the spore liquid is: from the isolated and purified I. javanica BE01 bacterial strain, pick mycelium and inoculate it on the SDAY plate covered with cellophane , cultivated at 25°C for 20 days, scraped the mycelium and spores on the cellophane and put them into 0.1% Tween-80 aqueous solution, stirred and oscillated fully, filtered to obtain the spore mother liquor, and adjusted the spore concentration to 1.0× ¹⁰⁴ / mL~1.0×10 ⁸ cells/mL. 5. The application according to claim 3, characterized in that: the concentration of the spore liquid is 1.0×10 ⁶ spores/mL~1.0×10 ⁸ spores/mL. 6. The method according to claim 1, wherein I. javanica for preventing and controlling white moth, is characterized in that: said I. javanica BE01 is cultivated using Chapei's medium. 7 . The method according to claim 6 , characterized in that: the I. javanica BE01 for preventing and controlling the white moth is cultured in Chapei's medium for 13 days at 25° C. 7 .