CN108330071A - Have Strain of Beauveria bassiana and its control of insect application of insect gut infection effect - Google Patents

Abstract

The invention relates to a Beauveria bassiana strain with insect intestinal infection effect and its pest control application. Specifically, the present invention relates to Beauveria bassiana (Beauveria bassiana) or its spores, mycelia or fermentation products whose microorganism preservation number is CGMCC No. 13180, a composition comprising the strain or its spores, and a preparation method thereof, and their use in pest control. The bacterial strain and composition of the present invention uniquely play a synergistic effect in preventing and controlling pests (such as Lepidoptera and Diptera insects) through intestinal infection and double infection of intestinal body wall, and have broad application prospects.

Description

Beauveria bassiana strain with insect intestinal infection and its application in pest control

technical field

The invention belongs to the fields of biotechnology and pesticides. The present invention relates to a new insecticidal fungus strain with intestinal virulence and body wall infection and its application in pest control, in particular to a new strain of Beauveria bassiana infected by insect foregut and its application in agriculture and Applications in insect-borne pest control.

Background technique

Insects occupy a dominant population on the earth, and many pests pose a great threat to human survival and health, such as causing crop yield reduction, endangering forestry development, spreading diseases, etc. (Pedigo & Rice, 2014, Entomology and pest management ( Waveland Press).).

Lepidoptera pests are the order with the most agricultural and forestry pests, such as pine caterpillars, cutworms, and Spodoptera litura, etc., and a large amount of manpower, material and financial resources are required to be invested in the control of pests every year to reduce their harm. In addition, mosquito-borne diseases are a class of diseases that seriously threaten human health, such as malaria, dengue fever, Zika and yellow fever, which cause hundreds of millions of infections and millions of deaths every year. Given the lack of effective vaccines and growing drug resistance, the main way to control mosquito-borne diseases is to cut off their transmission routes, ie reduce the number of mosquitoes.

At present, pest control mainly relies on chemical control methods, but with the extensive use of chemical pesticides, it has caused great pollution to the human living environment, and the pesticide residues in agricultural by-products have brought great harm to human health. . Moreover, the problem of insecticide resistance is becoming more and more serious, which limits the application of chemical pesticides.

Compared with chemical control, biological control has the advantages of safety, effectiveness and durability. In recent years, more and more biological control methods are used for pest control, mainly using insect natural enemies, parasites or pathogenic bacteria to achieve long-term effective control of pests (Lorito, 2016, Annual Review of Phytopathology, 55) .

Among biopesticides, biocontrol fungal preparations are the most widely used. They have the advantages of wide host range, good safety, easy production and preparation, etc., and were first used as biopesticides for pest control. Since 1960, there are 171 fungal insecticide products used for biological control, mainly derived from: Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae and Isariafunosorosea (de Faria & Wraight, 2007, Biological Control, 43, 237-256.). At present, entomopathogenic fungi have been widely studied and applied worldwide as an important biological control method.

Beauveria bassiana and Metarhizium anisopliae have been developed into a variety of preparations for the control of agricultural and forestry pests, but the control effect in the field is greatly restricted by environmental factors. Especially in the summer when there are a large number of pest outbreaks, high temperature, strong ultraviolet rays and other factors will affect the survival rate of fungi and insecticidal virulence, often resulting in unstable control effects of fungal agents, which has become an important limiting factor affecting the promotion and application of fungal insecticides , has also become a major international technical problem (Arthurs, 2001, Entomologia Experimentalis et Applicata, 100, 69-76; Braga, 2001, Photochemistry and Photobiology, 73, 140-146; Jaronski, 2009, In The ecology of fungalentomopathogens (Springer), pp .159-185.).

So far, the process of entomopathogenic fungus body wall invasion host infection and insecticidal mechanism have been studied relatively clearly, usually through the body wall penetration into the hemocoel of host insects, thereby causing the death of the host (Ortiz-Urquiza & Keyhani, 2013, Insects , 4(3), 357-374.). The whole infection process mainly includes the following stages: conidia adhere to the body wall of the host insect, spore germination, hyphae penetrate the body wall, and mycelia enter the insect hemolymph to proliferate in large numbers. Finally, the insect host dies due to a large amount of nutrients being exhausted. die. The entire infection cycle is relatively long and is greatly affected by environmental factors, which is also an important factor for the unstable control effect of fungal insecticides.

Since the environment of the insect digestive tract is relatively stable and can avoid the influence of the surrounding climate conditions, the digestive tract becomes the main route of bacterial and viral infection. The insect gut is considered to be an important place for the interaction between pathogenic microorganisms and hosts, especially the important targets for viruses, bacteria and other invading insects (Soberón et al., 2007, Science, 318, 1640-1642). Bacteria or viruses enter the intestinal tract of insects through feeding, and the peritrophic membrane is an important barrier for bacterial intestinal infection of the host. Pathogenic bacteria usually synthesize chitinase to help them penetrate the peritrophic membrane (Kelkenberg, Odman-Naresh, Muthukrishnan, & Merzendorfer, 2015, Insect Biochemistry and Molecular Biology, 56, 21-28.), while secreting various enzymes And the toxin acts on the midgut epithelial cells, and finally enters the hemocoel of the insect, causing the death of the insect host (Richards and Goodrich-Blair, 2010, Applied and Environmental Microbiology, 76, 221-229.).

For a long time, many researchers have tried to find highly virulent pathogenic fungi that can infect insects through the gut. However, biocontrol fungi usually lack specific intestinal virulence factors and can only infect hosts through body wall contact. In recent years, with the continuous advancement of filamentous fungal gene manipulation technology, great progress has been made in the study of gene manipulation and genetic improvement of fungi. In particular, the enterotoxic protein VipAal of the Vip3A family secreted by Bacillus thuringiensis was introduced into the transgenic engineering strain of Beauveria bassiana, which obtained a certain intestinal insecticidal activity, which is a good example for the realization of dual-path infection of entomopathogenic fungi. (Qin et al., 2010, Applied and Environmental Microbiology, 76, 4611-4618.).

In summary, although entomopathogenic fungi are important biological insecticides, especially Beauveria bassiana and Metarhizium anisopliae have been developed into a variety of preparations for the control of agricultural and forestry pests, but given that a single insect The infection route of the body wall, its field control effect is greatly restricted by environmental factors, and it is also a technical bottleneck that limits the large-scale application of fungal insecticides and an international problem that needs to be solved urgently. So far, there are very few examples of entomopathogenic fungi infecting host insects through the intestinal tract, and the research on the specific molecular mechanism is still blank. In order to solve the limiting factors in the application of biocontrol fungi, finding new strains with enteric virulence will effectively expand the development and application of biocontrol fungi, which is also of great significance for biocontrol.

Contents of the invention

The present invention just provides a novel bacterial strain with intestinal toxicity.

In some aspects of the present invention, a Beauveria bassiana is provided, wherein the microorganism preservation number of the Beauveria bassiana is CGMCC No.13180.

In some embodiments of the present invention, the Beauveria bassiana has virulence for intestinal infection of insects.

In some embodiments of the present invention, the Beauveria bassiana performs enteric infection through the foregut of the insect.

In some embodiments of the present invention, the Beauveria bassiana has characteristics of enteric infection virulence to Lepidoptera and Diptera pests.

In other aspects of the present invention, there is provided an insect control composition comprising:

(a) Beauveria bassiana described herein and/or its spores, mycelia or fermentation products thereof;

(b) Agrochemically acceptable carriers and/or adjuvants.

In some embodiments of the present invention, the composition is a dosage form suitable for killing insects through the intestinal tract of insects or through the body wall of the intestinal tract of insects.

In some embodiments of the present invention, the composition is in the form of a control effect through insect ingestion.

In some embodiments of the invention, the composition performs enteric infection through the insect foregut.

In some embodiments of the present invention, the dosage form of the composition is selected from: solution, emulsifiable concentrate, liquid suspension, dry suspension, wettable powder, dry powder, granule, water, poisonous bait, and microcapsule.

In some embodiments of the present invention, the insects are selected from: Lepidoptera, Diptera or other insects, such as moth pests or butterfly pests, such as pine caterpillars, cutworms, spodoptera litura, peach borer , apple leaf roller, cotton bollworm, cabbage butterfly, diamondback moth, borer moth, wheat moth, potato tuber moth, sweet potato wheat moth; mosquitoes, flies, horseflies, gnats, such as Anopheles, Culex, Aedes, Housefly, flower fly, toilet fly, decay fly, stinging fly, tsetse fly, blowfly, hemp fly, mad fly, skin fly, stomach fly, tick fly, gadfly, chironomus, gnat, crab gnat, yellow gnat, Golden gnats, Amazonian gnats, spotted gnats, bright-breasted gnats, brown-footed gnats, and giant gnats; wherein, the other types of insects include but are not limited to: Orthoptera insects, Coleoptera insects, Isoptera insects, etc. , such as locusts, cockroaches, scarabs, longhorns and termites.

In some embodiments of the present invention, the carrier and/or adjuvant is selected from: solvents, wetting agents, dispersants, emulsifiers, stabilizers, adhesives, fillers, release control aids, synergists, Adhesives, spreading agents, anti-drift agents, safeners, antidotes, defoamers, spray aids, warning colors, attractants, antifreeze agents.

In some embodiments of the present invention, the composition further comprises one or more components selected from the group consisting of pesticides, plant growth regulators, fertilizers and soil conditioners.

In some aspects of the invention there is provided a method of producing a composition described herein, the method comprising:

culturing, propagating or fermenting Beauveria bassiana described herein to obtain Beauveria bassiana described herein, or spores, mycelia or fermentation products thereof;

The resulting product is mixed with an agrochemically acceptable carrier and/or adjuvant.

In some embodiments, the fermentation is selected from: liquid fermentation, solid state fermentation, and liquid-solid two-phase fermentation.

In some embodiments, the method further comprises isolating, purifying, identifying, concentrating and/or storing the cultured, multiplied or fermented product.

In some aspects of the present invention, a method of controlling pests is provided, the method comprising:

Beauveria bassiana described herein, or spores, mycelia or fermentation products thereof, or a composition as described herein is applied to a locus where a pest is or is expected to be infested.

In some embodiments of the present invention, the place where harmful insects appear or are expected to appear is selected from one or more of the following group: farmland, vegetable field, orchard, woods, pasture, warehouse, residence, water area.

In some embodiments of the present invention, the application is by sowing, spraying, pouring, fumigation, soil mixing, seed dressing, or brushing.

In some aspects of the present invention, use of the Beauveria bassiana described herein, or its spores, mycelia or fermentation products, or the composition described herein in pest control is also provided.

Those skilled in the art can make any combination of the foregoing technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings, wherein these illustrations are only for illustrating the embodiments of the present invention, and are not intended to limit the scope of the present invention.

Figure 1: Colony morphology of Beauveria bassiana Bb-bm01 on SDAY medium.

Figure 2: Oral feeding of Beauveria bassiana Bb-bm01 spore suspension with a micropipette.

Figure 3: Virulence determination of silkworms infected with Beauveria bassiana Bb-bm01 intestinal tract.

A: Mortality of silkworm fed orally with different amounts of Bb-bm01 spores;

B: Mortality of silkworms fed orally with Beauveria bassiana Bb-bm01, Bb2860 and Bb252;

C: Mortality rate of mature silkworm larvae (fifth instar, fourth day) infected with Beauveria bassiana Bb-bm01 intestinal tract, where the photo shows the mycelial growth of silkworm larvae treated with Bb-bm01 after pupation.

Figure 4: Mortality of soybeans infected with Beauveria bassiana Bb-bm01 enteric infection, where the photos show the hyphae in soybeans treated with Triton X-100 and three days after death by Bb-bm01 treatment grow.

Figure 5: Mortality of silkworms infected with body wall, intestinal tract, and co-infected body wall and intestinal tract by Beauveria bassiana Bb-bm01.

Figure 6: Mortality of Anopheles stephenii enterically infected with Beauveria bassiana Bb-bm01, where photographs show mycelial growth in Anopheles stepheniensis three days after death from Bb-bm01 treatment.

Figure 7: PAS staining microscope observation of foregut paraffin section after oral feeding of Beauveria bassiana Bb-bm01 spores to silkworm.

A: 200 times magnification; B: 400 times magnification.

Fig. 8: Comparison of the virulence of the body wall of Beauveria bassiana Bb-bm01, Bb2860 and Bb252 infected silkworm.

Detailed ways

The present invention provides a method against insects (such as agricultural and forestry pests and vector pests, especially Lepidoptera pests (such as lepidoptera pests that seriously harm crops and forests) and Diptera pests (such as spreading malaria, dengue fever and yellow fever) Beauveria bassiana new bacterial strain Bb-bm01 of the mosquito-borne disease such as disease) has intestinal tract and body wall dual route infection concurrently. The present invention also provides a kind of approach of Bb-bm01 enteric infection insect simultaneously parse.

The intestinal virulence test of Beauveria bassiana Bb-bm01 showed that Bb-bm01 has intestinal infection virulence to Lepidoptera pests and Diptera pests. The observation of tissue section staining proved that Bb-bm01 carried out a new route of intestinal infection through insect foregut. Moreover, the co-infection of host insects through the body wall and intestinal tract effectively improved the insecticidal virulence of Bb-bm01. Therefore, the Bb-bm01 of the present invention can be made into a biopesticide for pest control. Thereby, the product of insecticide for entomogenic fungi can be more durable and effective, and the use of chemical pesticides can be reduced, which has important application value.

New strain Bb-bm01 of Beauveria bassiana and its preservation information

The new strain Bb-bm01 of Beauveria bassiana of the present invention is a new strain of Beauveria bassiana that the inventor separates and purifies from Bombyx mori worm. It has been tested that the bacterial strain has the virulence of insect intestinal infection, thereby preventing and controlling pests through intestinal infection and double infection of intestinal body wall, and the synergistic insecticidal effect is formed between these two ways. Moreover, Bb-bm01 is superior to other Beauveria bassiana known or commonly used in the art only in terms of the virulence of body wall infection of insects.

The Bb-bm01 strain of the present invention has been sent to the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee (CGMCC) for microbial preservation on November 22, 2016. Its preservation number is CGMCC No.13180, and the identification result is survival.

Composition and its preparation

The present invention also provides a composition comprising the strain of the present invention, which can be used for the prevention and control of pests, comprising: Beauveria bassiana Bb-bm01 of the present invention, or its spores, mycelia or fermentation products thereof or extract; and an agrochemically acceptable carrier and/or adjuvant.

As used herein, the term "active substance of the present invention" or "active substance for controlling pests of the present invention" can be used interchangeably, and refers to Beauveria bassiana Bb-bm01 of the present invention, or its spores, mycelium or Fermentation products or their extracts.

The strains of the present invention can be cultivated, multiplied, fermented, collected, etc. by conventional methods in the art. For example, the bacterial strain of the present invention can be cultured and propagated in an appropriate medium, which can be liquid or solid medium, including but not limited to: SDAY, PDA, SDB. Appropriate carbon sources, nitrogen sources, trace elements and pH regulators can be added to the basal medium as needed.

Beauveria bassiana of the present invention can also produce different types of spores or their mixtures under different culture conditions, such as producing blastospores and conidia. Under liquid culture conditions, Beauveria bassiana readily produces blastospores. Blastospores have thin cell walls, poor stress resistance, and are easily inactivated. Under solid culture conditions, Beauveria bassiana produces conidia, which have thicker cell walls and are more biologically stable than blastospores under normal conditions. In general, the conidia of Beauveria bassiana are more resistant to adverse environments than blastospores, so their conidia are preferred for use in pest control. But under special conditions or specific control objects, blastospores can also be used.

For the fermentation process of Beauveria bassiana, methods such as liquid submerged fermentation, solid-state fermentation and liquid-solid two-phase fermentation can be used, wherein the liquid-solid two-phase fermentation method is preferably used. For the production of entomopathogenic fungi, liquid-solid two-phase fermentation refers to the rapid production of a large number of hyphae or blastospores through liquid fermentation, and then inoculated on solid nutrients or inert substrates for solid fermentation to produce the closest natural inoculum Morphology of aerial conidia. This process combines liquid submerged fermentation and solid-state fermentation, giving full play to the advantages of both, overcoming the disadvantages of poor environmental stability of blastospores produced by pure liquid fermentation, and maximizing the use of the advantages of aerial conidia produced on solid surfaces , while shortening the growth cycle of mycelia, the production method is simple, the raw materials are easy to obtain, and the energy consumption cost is low. Therefore, it is widely used in the production of conidia of entomopathogenic fungi.

Agrochemically acceptable carriers and/or adjuvants are also included in the composition of the present invention. As used herein, the term "pesticidally acceptable" ingredient is a material suitable for use in a pesticide without unduly adversely affecting the activity of the active substance. As used herein, the term "effective amount" refers to an amount capable of controlling target insects. "Agrochemically acceptable carrier and/or adjuvant" refers to natural or synthetic organic or inorganic substances to which the active ingredient is combined to facilitate the application of the active ingredient to plants. Thus, the carrier is generally inert and agriculturally acceptable, especially on the plants to be treated, and may be, for example, a solid (clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, etc.) Or liquid (water, alcohols, ketones, petroleum fractions, aromatics or alkanes, chlorinated hydrocarbons, liquefied gas, etc.). These carriers or excipients themselves are not essential active ingredients, and will not have excessive adverse effects on the activity of essential active substances.

Carriers and/or adjuvants in the composition of the present invention may include, but are not limited to: solvents, wetting agents, dispersants, emulsifiers, stabilizers, adhesives, fillers, controlled release aids, synergists, adhesives , Spreading agent, anti-drift agent, safener, antidote, defoamer, spray auxiliaries, warning color, attractant.

The compositions of the present invention may also contain one or more other active substances in addition to the active substances of the present invention. Such combinations can provide certain advantages, such as, but not limited to: synergistic effect on controlling pests, reducing pesticide application rates, reducing environmental hazards, improving operator safety, providing broad-spectrum control, enabling Toxicity safening of plants, improved tolerance of non-pest species such as mammals and fish, etc.

Other active substances that may be used in the compositions of the present invention include, but are not limited to, other pesticides, plant growth regulators, fertilizers, soil conditioners or other agricultural chemicals.

In some embodiments, the active substances of the present invention may be applied in combination with one or more other pesticides, including but not limited to: organophosphate insecticides such as chlorpyrifos, dianon, dimethoate, horse Lathion, methyl-605, and terbufos; pyrethroid insecticides such as: fenvalerate (fenvalerate), deltamethrin, fenpropathrin, cyfluthrin, cyfluthrin Valerothrin, bifenthrin, cypermethrin, soluble cyhalothrin, etofenproxil, S-fenvalerate, perbalmethrin, tefluthrin, cypermethrin, -cyfluthrin, and fluorine Promethrin; carbamate insecticides such as: aldicarb, carbaryl, carbofuran, and methomyl; organochlorine insecticides such as endosulfan, endrin, heptachlor, and lindane; benzene Formoylurea insecticides such as: diflubenzuron, trimeturon, chlorfluron, diflunuron, hexaflumuron, flufenuron, and lufenuron; and other insecticides such as: acetamiprid, Tetrachlorid mite, pyraflux-methyl, hexythiazox, spinosyn, and imidacloprid; nematocides, such as carbofuran, carbosulfan, aldicarb, fenamiphos, chloramidofos, thiamid, Chlorazophos, Thiamiphos.

In some embodiments, the active substances of the present invention can be used in combination with other substances such as one or more plant growth regulators, including, for example, imabadan, chlormequat, ethephon, Gibberellins, mepizone, defolate, tributadiamide, imabazole, paclobutrazol, uniconazole, DCPA, fused cyclic acid, and other plant growth regulators.

In some embodiments, the active substances of the present invention may be applied in combination with one or more soil conditioning agents including: organic matter such as humus, which promotes the retention of cationic plant nutrients in the soil; cationic A mixture of nutrients, such as complexes of calcium, magnesium, caustic potash, sodium, and hydrogen; or a composition of microorganisms that promote soil conditions favorable to plant growth. Such microbial compositions include, but are not limited to, Bacillus, Pseudomonas, Azotobacter, Azospirillum, Rhizobium and Geocyanobacterium.

In some embodiments, the active substances of the present invention may be applied in combination with one or more fertilizers. Said fertilizers include, but are not limited to: nitrogen fertilizers, such as ammonium sulfate, ammonium nitrate, and bone meal; phosphate fertilizers, such as superphosphate, triple superphosphate, ammonium sulfate, and diammonium sulfate; potassium fertilizers, such as caustic potash chloride, potassium sulfate, and potassium nitrate.

The compositions of the present invention can be in various forms suitable for preparation, storage and administration. The dosage form of the composition can be selected from: solution, emulsifiable concentrate, liquid suspension, dry suspension, wettable powder, dry powder, granule, water, poison bait, microcapsule. For example, the compositions of the present invention may be in liquid form, which may be in the form of concentrated compositions or dilute compositions which are readily sprayed onto the plants to be treated.

In some embodiments of the present invention, the composition of the present invention is a dosage form suitable for killing insects through the intestinal tract of insects or through the body wall of the intestinal tract of insects. In some embodiments of the present invention, the composition is in the form of a control effect through insect ingestion. In some embodiments of the invention, the composition infects via the intestinal tract of the insect.

Applications of strains and compositions

The bacterial strain and composition of the present invention can be used for the prevention and control of pests through intestinal infection or compound infection of intestinal body wall.

Insects that can be controlled by the strains or compositions of the present invention include, but are not limited to: Lepidoptera, Diptera or other types of insects, such as moth pests or butterfly pests, such as pine caterpillars, cutworms, spodoptera litura, peach borer , apple leaf roller, cotton bollworm, cabbage butterfly, diamondback moth, bag moth, thorn moth, large silk moth, ruler moth, borer moth, dead leaf moth, boat moth, American white moth, national pagoda tree moth, borer moth, wheat moth , potato tuber moth, sweet potato wheat moth, etc; Blowflies, hemp flies, mad flies, skin flies, stomach flies, lice flies, gadflies, chironomids, evil gnats, crab gnats, light yellow gnats, golden gnats, Amazon gnats, spotted gnats, bright-breasted gnats, brown-footed gnats Gnats, giant gnats, etc. Other types of insects include, but are not limited to: locusts, cockroaches, scarabs, longhorns, and termites.

The strain or composition of the present invention can be applied to places where pests are or are expected to be infested, so as to eliminate, reduce or prevent pest damage caused by the pests. For example, the strain or composition of the present invention can be applied to farmland (for example, areas where crops such as cereals, cotton, vegetables, and fruits grow or areas where these crops are to be grown), woods (for example, for forestry protection), pastures, warehouses, Residences, waters, etc. where necessary. The bacterial strain or composition of the present invention can also be used in the field of veterinary medicine, for example, to effectively control internal and external parasites that plague animals, such as insect nuisances such as mosquitoes, flies, and horseflies. .

The application methods of the strain or composition of the present invention can be sowing, spraying, pouring, fumigation, soil mixing, seed dressing, brushing and the like. Those of ordinary skill in the art can select appropriate formulation forms and corresponding administration methods according to the needs in practical applications.

In addition, the strain or composition of the present invention can also be applied in combination with other active substances as needed to provide additional advantages, such as but not limited to: synergistic effect on pest control, reduced application rate of pesticides, reduced Environmental hazards, enhance operator safety, provide broad spectrum control, make toxic to plants safe, improve tolerance of non-pest species such as mammals and fish, etc. Other active substances that may be applied in combination with the strains or compositions of the invention include, but are not limited to: other pesticides, plant growth regulators, fertilizers, soil conditioners or other agrochemicals (as described above). The active substances according to the invention and the other active substances can be administered simultaneously, sequentially or alternately.

All numerical ranges provided herein are intended to expressly include all values falling between the endpoints of the range and numerical ranges therebetween. Features mentioned in the invention or features mentioned in the embodiments may be combined. All the features disclosed in this specification can be used in combination with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

As used herein, "comprising", "having" or "comprising" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of Consists of ", "essentially composed of" and "consisting of" belong to the sub-concepts of "contain", "have" or "include".

Described herein are only a few embodiments of the present invention. Those skilled in the art can make various combinations, changes or modifications to the present invention without departing from the spirit and concept of the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are all within the scope of the present invention.

For the experimental methods that do not indicate specific conditions in the following examples, conventional methods in the art can be used, for example, with reference to "Molecular Cloning Experiment Guide" (third edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or as suggested by the supplier. The DNA sequencing method is a routine method in the art, and commercial companies can also provide tests.

Percentages and parts are by weight unless otherwise indicated. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1. Isolation and Identification of Bacterial Strain Bb-bm01

The bacterial strain Bb-bm01 of the present invention is a new bacterial strain of Beauveria bassiana isolated and purified from the silkworm worm. Bb-bm01 was specifically screened by the following method: under sterile conditions, put the silkworm covered with white hyphae all over the body on the sterilized SDAY medium plate and shake gently, and then place the SDAY plate at 27°C for cultivation Cultivate in an incubator for 3 to 4 days, pick a single colony to a purification medium (SDAY), and carry out purification culture in a 27°C incubator. It was found that a strain similar to Beauveria bassiana had a velveteen-like round colony on SDAY medium, the color gradually changed from white to milky white or light yellow, the hyphae were fluffy, and the powder layer was loose, and it was named Bb-bm01 (figure 1).

Bb-bm01 was sent to the General Microorganism Center (CGMCC) of China Committee for Microbial Culture Collection on November 22, 2016 for microbial preservation. Its preservation number is CGMCC No.13180, and the identification result is survival.

The hyphae of strain Bb-bm01 were picked from SDAY medium for total DNA extraction, the sequence of ITS (internal transcribed spacer, internal transcribed spacer) was amplified by PCR, and Bb-bm01 was identified by sequence alignment. The specific primers for PCR amplification are:

ITS-F (SEQ ID NO: 1): 5'-TCCGTAGGTGAACCTGCTGAGGGAT-3';

ITS-R (SEQ ID NO: 2): 5'-TCCTCCGCTTATTGATATGCTTAA-3'.

The length of the amplified Bb-bm01 ribosomal DNA-ITS was 569bp; the PCR product was cloned into the pMD-18T vector (Takara), and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The DNA sequence obtained by sequencing is (SEQID NO:3):

TCCGTAGGTGAACCTGCTGAGGGATCATTACCGAGTTTTCAACTCCCTAACCCTTCTGTGAACCTACCTATCGTTGCTTCGGCGGACTCGCCCCAGCCCGGACGCGGACTGGACCAGCGGCCCGCCGGGGACCTCAAACTCTTGTATTCCAGCATCTTCTGAATACGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAACGCGATAAGTAATGTGAATTGCAGAATCCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACCTCCCCTTGGGGAGGTCGGCGTTGGGGACCGGCAGCACACCGCCGGCCCTGAAATGGAGTGGCGGCCCGTCCGCGGCGACCTCTGCGCAGTAATACAGCTCGCACCGGGACCCCGACGCGGCCACGCCGTAAAACACCCAACTTCTGAACGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA

Comparing the sequencing results with the NCBI gene database shows that the ribosomal DNA-ITS sequence of Bb-bm01 is as high as 99% homologous to the ribosomal DNA-ITS sequence of multiple strains of Beauveria bassiana in the NCBI database, which indicates that Bb - bm01 is Beauveria bassiana.

At the same time, the determination and analysis of the ITS sequence further proved that Bb-bm01 is different from the known strains of Beauveria bassiana.

Embodiment 2. The method of feeding insect Beauveria bassiana spores orally

The invention establishes a method for orally feeding the insect Beauveria bassiana. Take the silkworm, a model animal of Lepidoptera, as an example:

Prepare a certain concentration of fresh Beauveria bassiana spore suspension with 0.01% Triton X-100 sterile water. The silkworm larvae on the second day of the fifth instar were selected. The head of the larva was fixed, and 1 μl of the spore suspension was aspirated with a micropipette (Gilson, P2), and slowly injected into the mouth of the silkworm larvae (Fig. 2). The whole process prevents the spore suspension from contacting the mouthparts and body wall of the larvae, and prevents the spores of Beauveria bassiana from infecting insects through the body wall. Then it is placed in an incubator with a temperature of 27°C and a humidity of 30-40%, and is fed with fresh mulberry leaves.

Example 3. Beauveria bassiana Bb-bm01 enteric infection of Lepidoptera larvae

Inoculate Beauveria bassiana Bb-bm01 on SDAY medium, culture in 27°C incubator for 10 days, then prepare conidia suspension with 0.01% Triton X-100 sterile water, and measure spores with hemocytometer Concentrations were formulated into spore suspensions at concentrations of 1×10 ⁹ , 1×10 ⁸ , 1×10 ⁷ , 1×10 ⁶ , 1×10 ⁵ , and 1×10 ⁴ spores/ml (conidia/ml).

A. Targeting the second day larvae of the fifth instar of silkworm P50 strain

The second-instar larvae of the fifth instar silkworm P50 strain were used to test the intestinal toxicity of Beauveria bassiana Bb-bm01. The feeding conditions of the silkworm larvae were 27°C and 40% humidity. Take 1 μl of different concentrations of Bb-bm01 spore suspension, and use a micropipette to orally feed the second-instar larvae of the fifth instar silkworm P50, and the blank control is 0.01% Triton X-100. There were 30 silkworm larvae in each group, and each concentration was replicated three times, and the death of larvae was recorded every day. The results of intestinal toxicity test showed that the strain Bb-bm01 was enteropathogenic to silkworm larvae. When each silkworm was fed with 10,000 spores, all the silkworm larvae died after five days, while the silkworm larvae in the control group did not die (Fig. 3A) .

At the same time, we also tested the intestinal virulence of two other strains of Beauveria bassiana commonly used in the laboratory, Bb2860 and Bb252 (both purchased from ARSEF, the United States Department of Agriculture Collection of Entomopathogenic Fungi), and the feeding conditions of silkworm larvae were as above. Take 1 μl of the spore suspensions of Bb-bm01, Bb2860 and Bb252 with a concentration of 1× ¹⁰⁸ spores/ml, and feed them orally to the second-instar larvae of the fifth instar silkworm P50 with a micropipette (Gilson, P2), and the blank control 0.01% Triton X-100. There were 30 silkworm larvae in each group, and each concentration was replicated three times, and the death of larvae was recorded every day. The results of intestinal virulence assay showed that neither the strains Bb2860 nor Bb252 had enteropathogenicity to silkworm larvae (Fig. 3B).

B. For the mature larvae of silkworm (the fifth instar and fourth day larvae of silkworm P50)

In addition, 1 μl concentration of 1× ¹⁰⁷ spores/ml Bb-bm01 was used to orally feed mature silkworm larvae (larvae of the fifth instar and fourth day of silkworm P50 strain), and the control group was 0.01% Triton X-100, There were 30 silkworm larvae in each group, and three groups were repeated. The results of virulence assay showed that Bb-bm01 also had enteric infection virulence to mature larvae, and all the silkworms died after 6 days of infection (Fig. 3B); The head of the pupa begins to grow, and then slowly grows over the whole body (Fig. 3C).

C. Aiming at Dandan (bean hornworm larvae)

Taking the Lepidoptera pest Clanis bilineata as the research object, the third-instar Clanis bilineata (Hermus larvae larvae) was selected to test the intestinal toxicity of Beauveria bassiana Bb-bm01. , humidity 30-40%. Take 1 μl of Bb-bm01 spore suspension (1×10 ⁹ spores/ml), and use a micropipette to orally feed Dou Dan, the blank control is 0.01% Triton X-100, 30 heads of Dou Dan in each group, three for each Repeat, recording Doudan deaths every day. The measurement results showed that the strain Bb-bm01 had intestinal infection virulence to the lepidopteran pest Dandan, and after the death of Dandan, the mycelia of Bb-bm01 gradually penetrated the body wall of Dandan and grew in large quantities on its body surface (Figure 4).

Conclusion: The above results prove that the bacterial strain Bb-bm01 of the present invention has a control effect on Lepidoptera insects, and can play a role through intestinal infection.

Example 4. Beauveria bassiana Bb-bm01 body wall and intestinal co-infection have insecticidal synergistic effect

Taking the second-instar larvae of the fifth instar silkworm P50 strain, a model animal of Lepidoptera, as the research object, the insecticidal virulence of body wall infection of Beauveria bassiana Bb-bm01 was measured by spraying. The concentration of Bb-bm01 spore suspension is 1× ¹⁰⁷ spores/ml, the control group is 0.01% Triton X-100, 30 silkworm larvae in each group are sprayed with 5ml respectively, and placed in a room with a temperature of 27°C and a humidity of 90%. Breeding in an incubator, repeated in three groups. The results showed that Bb-bm01 also had high pathogenicity to silkworm larvae through body wall infection (Figure 5).

At the same time, Bb-bm01 was used to infect the body wall, intestinal tract and combined infection of the body wall and intestinal tract on the fifth instar larvae of silkworm P50, respectively. Among them, the concentration of Bb-bm01 spore suspension for body wall infection was 1×10 ⁷ spores/ml, the number of Bb-bm01 spores/head for intestinal infection silkworm larvae was 10 ⁴ spores/head, and the blank control was 0.01% Triton X -100. There were 30 heads in each group, three replicates each, and the death of silkworms was recorded every day.

The results of the virulence assay showed that the virulence of the co-infection of the body wall and intestinal tract of Beauveria bassiana Bb-bm01 was significantly higher than that of a single infection of the body wall (p<0.001, Log-rank (Mantel-cox) Test) or intestinal infection Infection (p<0.0001, Log-rank (Mantel-cox) Test) (Figure 5). The results proved that dual-path co-infection can effectively improve the insecticidal virulence of the strain Bb-bm01, and has synergistic insecticidal effect.

Embodiment 5. Beauveria bassiana Bb-bm01 has effect on Diptera pest Anopheles stephensi (Anopheles stephensi) Enteric virulence

Prepare fresh Bb-bm01 spore suspension with 0.01% Triton X-100 sterile water, measure the spore concentration with a hemocytometer, and prepare a spore suspension with a concentration of 1×10 ⁸ spores/ml, and then mix with sterile 10 % sucrose water mixed in equal volume. Adults of Anopheles stepheni, a dipteran pest, were used to test the virulence of intestinal infection of Beauveria bassiana Bb-bm01. The breeding conditions of Anopheles stephenii were temperature of 26° C. and humidity of 90%. Soak sterile cotton balls with 10% sucrose water containing 5× ¹⁰⁷ spores/ml Bb-bm01 spores, place the cotton balls containing spores on the top of the mosquito breeding container for Anopheles stephensi to eat; the blank control is containing 0.01% Triton X-100 in 10% sucrose water, 50 adult Anopheles stephensi in each group, three replicates each, and the death of mosquitoes was recorded every day.

The measurement results showed that the strain Bb-bm01 had enteric infection virulence to the Diptera pest Anopheles stephenii, and the hyphae of Bb-bm01 penetrated through the head of the mosquito after the mosquito died ( FIG. 6 ). The above results show that the bacterial strain Bb-bm01 of the present invention can effectively control Diptera pests, and the control can be carried out through intestinal infection.

Example 6. Beauveria bassiana Bb-bm01 carries out enteric infection through the foregut of silkworm

Another important problem to be solved by the present invention is to clarify the specific site and route of intestinal infection of host insects by Beauveria bassiana Bb-bm01.

Prepare fresh Bb-bm01 spore suspension with 0.01% Triton X-100 sterile water, measure the spore concentration with a hemocytometer, and prepare a spore suspension with a concentration of 1×10 ⁷ spores/ml. Use a micropipette to take 1 μl of Bb-bm01 spore suspension (1×10 ⁷ spores/ml) and feed it to the fifth-instar and second-instar larvae of the silkworm P50 strain, and the control group is Beauveria bassiana without intestinal infection virulence Bb252 (laboratory-preserved strain, tested to have no enteric infection virulence) spore suspension (1×10 ⁷ spores/ml). The breeding conditions of the silkworm larvae are temperature of 26° C. and humidity of 30-40%.

The silkworm larvae 12h, 24h, 48h and 84h after feeding the spores were taken respectively, and the intestinal tract of the infected silkworm was dissected and separated immediately, and placed in a fixative solution (60% absolute ethanol, 30% chloroform, 10% glacial acetic acid), 4 ℃ for 24h. After fixation, rinse twice with 70% ethanol, and finally store in 70% ethanol. Then the tissue was transferred to 100% absolute ethanol, put in a mixer and rotated for 1 hour for dehydration (3 times). Place the dehydrated intestinal tissue in xylene for 15 min (3 times). The intestinal tissues were put into melted paraffin at 65°C and embedded for 2 days. The embedded samples were left at room temperature for 2 hours, then at 4°C for 20 minutes, and cut into thin slices with a thickness of 5 μm with a microtome. Put the sliced slices on a gelatin-coated glass slide, place them on a rotator (42°C) and bake them for 15 minutes. After the slices are flattened and absorb excess gelatin, place them on the rotator for 24h-36h. Sections were then soaked in xylene for 20 minutes to dewax. The dewaxed samples were rehydrated, washed with 100%, 95%, 80%, and 70% ethanol for 5 minutes each time, and ddH ₂ O for 3 minutes (twice). Finally, the sections were stained with PAS and observed under a microscope after staining.

The results showed that Bb-bm01 was orally infected through the foregut of silkworm. After feeding the silkworm with Bb-bm01, the spores of Bb-bm01 adhered to the folds of the foregut of the silkworm, germinated, penetrated the foregut wall and entered the hemocoel of insects to form thalli, and multiplied in large numbers in the hemocoel of insects (Figure 7). At the same time, we also made paraffin sections of silkworms fed with the control strain Bb252, which had no enteric infection ability. The results showed that the spores of Bb252 did not germinate in the silkworm gut. As time went by, the spores of Bb252 passed through the foregut, midgut and hindgut in turn, and finally all were excreted from the body.

Embodiment 7. Beauveria bassiana Bb-bm01, Bb2860 and Bb252 body wall infection silkworm virulence comparison

The second-instar larvae of the fifth instar silkworm P50 strain were used to measure the insecticidal virulence of body wall infection against Beauveria bassiana Bb-bm01, Bb2860 and Bb252 by spraying. The breeding conditions of the silkworm larvae are temperature 27° C. and humidity 40%. Use 0.01% Triton X-100 to collect fresh Beauveria bassiana Bb-bm01, Bb2860 and Bb252 respectively, the concentration of spore suspension is 1× ¹⁰⁷ spores/ml, and the control group is 0.01% Triton X-100 . Each group of 30 silkworm larvae was sprayed with 5ml respectively, placed in an incubator with a temperature of 27° C. and a humidity of 90% to raise them, repeated in three groups, and recorded the death of the larvae every day.

The results of virulence assay showed that: compared with the other two strains of Beauveria bassiana Bb2860 and Bb252, Bb-bm01 also had high pathogenicity to silkworm larvae through body wall infection (Fig. 8).

It should be understood that what is listed above are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, Bb-bm01 can also infect and thus control other insects, such as Spodoptera litura (Lepidoptera), Corn borer (Lepidoptera), locusts, cockroaches (Orthoptera), Scarabs, beetles (Coleoptera), termites (Isoptera), etc.

In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Beauveria bassiana strain with insect intestinal infection and its application in pest control

<130> 167998 1CNCN

<160> 3

<170> PatentIn version 3.3

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<213> Beauveria bassiana

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Claims (10)

1. A Beauveria bassiana (Beauveria bassiana), wherein the microorganism preservation number of the Beauveria bassiana is CGMCC No.13180. 2. An insect control composition comprising: (a) Beauveria bassiana as claimed in claim 1 and/or its spore, mycelium or its fermentation product; (b) Agrochemically acceptable carriers and/or adjuvants. 3. The composition according to claim 2, wherein the composition is a dosage form suitable for killing insects through the intestinal tract of insects or through the body wall of the intestinal tract of insects. 4. The composition as claimed in claim 2, wherein said insect is selected from the group consisting of: Lepidoptera, Diptera or other classes of insects, such as moth pests or butterfly pests, such as pine caterpillars, cutworms, nightworms Moths, peach borer, apple leaf roller, cotton bollworm, cabbage butterfly, diamondback moth, borer moth, wheat moth, potato tuber moth, sweet potato wheat moth; mosquitoes, flies, horseflies, gnats, such as Anopheles, library Mosquitoes, Aedes mosquitoes, houseflies, flower flies, toilet flies, decay flies, biting flies, tsetse flies, blowflies, hemp flies, mad flies, skin flies, stomach flies, lice flies, gadflies, chironomids, evil gnats, crab gnats , light yellow gnat, golden gnat, Amazon gnat, spotted gnat, bright-breasted gnat, brown-footed gnat, and giant gnat; wherein, the other types of insects include but are not limited to: Orthoptera insects, Coleoptera insects, Isoptera insects, such as locusts, cockroaches, scarabs, longhorns and termites. 5. composition as claimed in claim 2, described carrier and/or adjuvant are selected from: solvent, wetting agent, dispersant, emulsifier, stabilizer, adhesive agent, filler, controlled release aid, enhancer Effective agents, adhesives, spreading agents, anti-drift agents, safeners, antidotes, defoamers, spray aids, warning colors, attractants, antifreeze agents. 6. The composition of claim 2, further comprising one or more components selected from the group consisting of pesticides, plant growth regulators, fertilizers and soil conditioners. 7. A method of producing a composition as claimed in any one of claims 2 to 6, said method comprising: Cultivate, proliferate or ferment the Beauveria bassiana described in claim 1, to obtain the Beauveria bassiana as claimed in claim 1 or its spore, mycelium or fermentation product; The resulting product is mixed with an agrochemically acceptable carrier and/or adjuvant. 8. A method for controlling pests, said method comprising: Beauveria bassiana as claimed in claim 1, or its spores, mycelium or fermentation product, or the composition as described in any one of claims 2 to 6 is applied to harmful insects or expected harmful insects haunted place. 9. The method according to claim 8, the place where the pests appear or are expected to appear is selected from one or more of the following group: farmland, vegetable field, orchard, woods, pasture, warehouse, residence, waters. 10. The application of Beauveria bassiana as claimed in claim 1, or its spores, mycelium or fermentation product, or the composition as described in any one of claims 2-6 in pest control.