CN113308385A - Isaria javanicus strain DMC01 and application thereof in prevention and treatment of locusta migratoria in east Asia - Google Patents

本发明属于微生物学技术领域，具体涉及的是一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用。一种爪哇棒束孢菌株DMC01，其分类学名为Isaria javanica，于2021年4月28日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC No.61632，其基因序列如SEQ ID No.3所示。本发明的爪哇棒束孢DMC01对东亚飞蝗具有较高致病力，可有效杀死东亚飞蝗，且具有较高僵虫率。本发明的爪哇棒束孢DMC01是一株在东亚飞蝗防治上具有潜在意义的生物防治菌株。

The invention belongs to the technical field of microbiology, and specifically relates to a Corynebacterium javanica strain DMC01 and its application in preventing and controlling the East Asian locust. A Corynebacterium javanica strain DMC01, whose taxonomic name is Isaria javanica, was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 28, 2021, and the deposit number is GDMCC No.61632, and its gene sequence is such as SEQ ID No. 3 shown. The Corynebacteria javanica DMC01 of the present invention has high pathogenicity to East Asian migratory locusts, can effectively kill East Asian migratory locusts, and has a higher rate of mussels. The Corynebacteria javanica DMC01 of the present invention is a biological control strain with potential significance in the control of East Asian locusts.

Isaria javanicus strain DMC01 and application thereof in prevention and treatment of locusta migratoria in east Asia

Technical Field

The invention belongs to the technical field of microbiology, and particularly relates to an Isaria javanicus strain DMC01 and application thereof in prevention and treatment of locusta migratoria in east Asia.

Background

Locusta migratoria Locusta (Meyen) belong to the class entomophyceae, order orthoptera, family locustaceae, namely locust and locust. In china, locusta migratoria in east asia is widely distributed in north China, east China, southwest China, south China, Taiwan and the like, while the yellow-Huai-Hai region is a common area. The locusta migratoria is a big migratory and omnivorous pest, and mainly eats grassy, cyperaceae and other weeds and crops, wherein the crops eat corns, paddy rice, sugarcane and the like, so that great harm is brought to agricultural production in various regions. The migratory locust in east Asia cannot be harmed from the physiological and ecological characteristics of high propagation speed, large number of reproductive offspring, strong migratory flight, wide feeding range, large food intake and the like. At present, measures for preventing and controlling locust disasters at home and abroad comprise chemical prevention and control, ecological prevention and control and biological prevention and control means, wherein the chemical prevention and control means are taken as main prevention and control means. Although chemical control has the advantages of high efficiency, quick effect, convenient use, high economic benefit and the like, the chemical control has the disadvantages of environmental pollution, natural enemy killing, harm to human and livestock, easy pest resurgence and the like.

The locusts have a plurality of natural enemies, such as predatory insects, such as turnip, bee fly and the like, predatory birds, such as lark, sparrow and pink, 26891and birds, but the mature technology and the efficient technology for preventing and treating the locusts by utilizing the natural enemies of the locusts are not many at present. In the prior art, the use of entomogenous fungi such as Beauveria bassiana (Beauveria bassiana) and Metarhizium anisopliae (Metarhizium) for controlling locusta migratoria in east Asia has been reported. At present, no report related to the use of Isaria javanicus (Isaria japonica) for controlling locusta migratoria in east Asia exists.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide an Isaria javanicus strain DMC01 and application thereof in controlling locusta migratoria in east Asia.

Isaria javanicus strain DMC01, the classification science of which is Isaria java vanica, and the gene sequence of which is shown as SEQ ID No. 3.

The Isaria javanicus strain DMC01 provided by the invention is obtained by collecting natural infected insects, separating and purifying, is identified as Isaria javanicus by morphology and molecular biology, is preserved in the Guangdong province microbial strain preservation center in 28 th 2021 (GDMCC for short, the address: No. 59 building 5 of Mr. shod 100 of Guangzhou city, and the address: the postcode: 510070 of Guangdong province microbial research institute), and has the preservation number of GDMCC No. 61632.

The invention also provides application of the Isaria javanicus strain DMC01 in prevention and treatment of locusta migratoria in east Asia, and technical personnel in the technical field can prepare Isaria javanicus strain DMC01 into a microbial insecticide for prevention and treatment of locusta migratoria in east Asia.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method separates and purifies the strain, and determines the obtained strain DCM01 as Isaria javanica through morphological observation and molecular identification; secondly, the applicant also determines the toxicity of the Isaria javanicus DMC01 on the east Asia migratory locust, and the Isaria javanicus DMC01 has high pathogenicity on the east Asia migratory locust, can effectively kill the east Asia migratory locust and has high rate of stiff insects.

(2) The Isaria javanicus DMC01 is a biological control strain with potential significance in controlling locusta migratoria in east Asia, can be developed into a microbial insecticide, and provides technical support for green control of locusta migratoria in east Asia.

Description of preservation information

Isaria japonica DMC01 with the preservation number of GDMCC No.61632, the preservation date of 2021, 4 months and 28 days, the preservation unit is Guangdong provincial microorganism culture Collection (GDMCC), and the preservation address is No. 59, 5 th of Michelia Tokyo No. 100 college in Guangzhou city.

Drawings

FIG. 1 is a front view of the colony morphology of DMC01 strain;

FIG. 2 is a back view of the colony morphology of DMC01 strain;

FIG. 3 is a phylogenetic diagram of the DMC01 strain;

FIG. 4 shows the infestation of DMC01 strain on migratory locusts in east Asia;

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The materials and reagents used in the following examples are commercially available, unless otherwise specified. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Examples

Separation and purification of Isaria javanicus strain DMC01

Collecting dead insects of eucalyptus caterpillar in Qincui forest farm of Qinzhou, Guangxi Zhuang, on a super clean bench, immersing the dead insects in 75% alcohol for surface disinfection for 30s, soaking and disinfecting for 3min by using 0.1% mercuric chloride, then sequentially transferring to 3 bottles of sterile water for washing, taking out and placing on sterilized filter paper to dissect the insects into small blocks;

placing the tissue blocks on PDA culture medium added with 50ug/mL streptomycin, placing 5 blocks per dish, culturing at 25 deg.C, picking out the transferred strain after hypha grows out, placing on PDA flat plate for purification culture, and repeating the purification culture for 3 times. The number of the screened fungus is DMC01, hypha obtained by purification is selected and inoculated into a PDA slant test tube, and the hypha is placed in an environment of 4 ℃ for preservation after the slant test tube is full of the fungus.

II, identifying Isaria javanicus strain DMC01

The bacterial colonies were observed during the culture of the strains cultured at 25 ℃ and the morphology of the colonies is shown in FIGS. 1 and 2.

As can be seen from FIGS. 1 and 2, on the PDA plate, the colony was round, white in hyphae, thick fluffy in texture, pale yellow on the back of the colony, and light purple gray in color of the spore layer.

Under a microscope, hyphae are colorless and transparent, a rotabiosome formed by 2-4 bottle stalks is grown on the molecular spore stalks, and the base parts of the bottle stalks are expanded in an oval shape and gradually thinned upwards; conidia grow on the phialides to form spore chains; conidia are transparent and smooth in oblong shape.

According to the characteristics of colony morphology, spore morphology and the like, the strain is preliminarily identified as Isaria javanica.

III, molecular identification

1. DNA was extracted according to the instructions of the Nuclean Plant Genomic DNA Kit of Kangji century to obtain Genomic DNA of Isaria javanicus strain DMC 01.

2. Amplifying a target gene beta-tubulin (beta-tubulin, TUB2) by adopting fungus TUB primers Bt2a and Bt2b to obtain a PCR amplification product; the sequences of the primers Bt2a and Bt2b are shown in Table 1, and the PCR reaction system is shown in Table 2.

TABLE 1 sequences of primers Bt2a and Bt2b

TABLE 2 PCR reaction System

PCR reaction procedure: denaturation at 95 deg.C for 3 min; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 10 min.

3. And recovering and purifying the amplification product, connecting and converting the amplification product, and performing DNA sequencing to obtain a nucleotide sequence shown in SEQ ID No. 3.

4. The nucleotide sequence was BLAST aligned to known sequences in NCBI to construct a phylogenetic tree of the DMC01 strain, the results of which are shown in FIG. 3.

As can be seen from FIG. 3, the DMC01 strain was clustered with AY624224(Isaria japonica) and its morphological characteristics were in accordance with those of Isaria javanicus, and it was determined that strain DMC01 was Isaria javanicus. Therefore, it was named Isaria java DMC 01.

Virulence determination of Isaria javanicus strain DMC01 on locusta migratoria

1. Scraping conidia of Isaria javanica strain obtained by separation and purification into 50mL triangular flask with inoculating loop, adding 20mL Tween-80 aqueous solution containing 0.05%, shaking thoroughly, counting with blood counting plate under microscope to obtain initial concentration spore mother liquor, preparing spore solution into 1 × 10⁵、1×10⁶、1×10⁷、1×10⁸Each concentration is one treatment, and each treatment is provided with 3 groups of repetitions by taking 0.05% Tween-80 aqueous solution as a control, and each repetition is 15 migratory locusts in east Asia.

2. Uniformly spraying prepared spore solutions with various concentrations and a 0.05% Tween-80 aqueous solution subjected to control treatment on a migratory locust in east Asia until the spore solutions do not drip, then placing the migratory locust in east Asia into a pest-culturing dish by a single head, culturing in an incubator at 25 ℃ for 12h in light and dark, feeding with fresh corn leaves every day, regularly observing and recording the specific death condition of each treatment every day, continuously observing for 8d, removing the culture dish when dead pests are found, placing the dead pests into a glass culture dish with filter paper and sterilized, and performing wet culture to observe whether hyphae grow out, wherein the result is shown in figure 4.

As can be seen from FIG. 4, hyphae were grown on the planthopper after the DMC01 strain had infested the migratory locust in east Asia.

The number of heads of the dead insects was recorded, and the corrected mortality rate of different concentrations of Isaria javanicus DMC01 on four-year-old eastern Asian migratory locusts and the dead insect rate of four-year-old eastern Asian migratory locusts after different concentrations of Isaria javanicus DMC01 treatment were calculated, and the results are shown in tables 3 and 4 below.

TABLE 3 corrected mortality of Isaria javanicus DMC01 at various concentrations on fourth-instar locusta migratoria

As can be seen from table 3, the higher the spore concentration, the higher the corrected mortality rate for locusta migratoria in east asia; at the lowest concentration of 1X 10⁵The corrected mortality rate of the strain after 8 days of inoculation at one/mL also reaches 69.05%; with followingThe corrected mortality rate of locusta migratoria in east asia is increased by the extension of the inoculation time, and the corrected mortality rate of the inoculated locusta migratoria 8d is higher than that of the inoculated locusta 4d under the treatment of the same spore concentration.

TABLE 4. varying concentration Isaria javanicus DMC01 treatment for Metaplexis migratoria Selenii Selenz rate

As can be seen from Table 4, the migratory locust in east Asia treated with Isaria javanicus DMC01 has high rate of dead insects, with the lowest concentration of 1X 10⁵The rate of Bombyx Batryticatus under one/mL treatment was 44.44%, compared with 1X 10⁷one/mL and 1X 10⁸The stiff worm rate under the condition of each/mL treatment is 100 percent.

As can be seen from the combination of tables 3 and 4, Isaria javanicus DMC01 of the present invention is highly pathogenic, 1X 10, to locusta migratoria⁷Spore liquid with concentration more than one/mL can effectively kill locusta migratoria in east Asia, and has high rate of dead insects.

In conclusion, the Isaria javanicus DMC01 has high pathogenicity on the locusta migratoria in east Asia, can effectively kill the locusta migratoria in east Asia, has high muscardine rate, and is a biological control strain with potential significance in the control of the locusta migratoria in east Asia.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Sequence listing

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