A simple method for inducing appressorium of Metarhizium anisopliae

Abstract

The present invention discloses a simple induction method for appressoria of Metarhizium rileyi, which comprises the following steps: S1: Weigh agar powder and add 150 mL of pure water, mix evenly and sterilize at 121 °C; S2: Sterilize qualitative filter paper at high temperature, and evenly cut it into 4 equal parts in a super clean bench, and spread the filter paper pieces on the petri dish in step S1; S3: Sterilize square cover glasses at high temperature, and carefully place the cover glasses on the surface of the filter paper in step S2; S4: Transfer and culture the primary Metarhizium rileyi strain for 7 - 10 days until a large amount of spores are produced, and use 0.05% Tween 80 as a solvent to prepare a conidia suspension with a concentration of 1x10<supgt;6< / supgt> / mL for standby; S5: Pipette 50 μL of the conidia suspension in step S4, drop it on the upper surface of the cover glass in step S3, and smear and distribute it throughout the glass slide to obtain a prepared petri dish; S6: Carefully seal the petri dish in step S5, and observe the formation of appressoria under a microscope after cultivation. The present invention provides a theoretical and technical means for creating highly virulent Metarhizium rileyi strains.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a simple induction method for Metarhizium anisopliae appressorium. Background Art

[0002] Metarhizium rapa is an entomogenous fungus of the genus Metarhizium and is a widely distributed insect pathogenic fungus in the world. Under natural field conditions, it can infect more than 40 species of insects in the Noctuidae and other families of the Lepidoptera order, causing large-scale infectious diseases within the population. It has great research value and application prospects. The exploration of the infection mechanism of entomogenous fungi in biological control and the exploration of means to improve the toxicity of entomogenous fungi will greatly promote the development of green agricultural production. Metarhizium rapa mainly invades the body wall of pests by producing special structures such as appressorium and germ tubes, and then grows and reproduces rapidly in the insect body, destroying the physiological functions of the pests and ultimately causing the death of the pests.

[0003] At present, the most widely used and studied entomogenous fungi include Beauveria bassiana, Metarhizium anisopliae, Metarhizium muscardineum, etc. These entomogenous fungi have a wide range of hosts, including many insects such as Lepidoptera, Coleoptera, and Orthoptera. There are also many reports on the methods of inducing appressorium of these entomogenous fungi. There are two commonly used induction media: 1. Insect hind wings. Inoculate with the hind wings of insects such as locusts as the matrix, and observe the formation of appressorium under a microscope; 2. YE (Yeast Extract) medium. By measuring the formation of appressorium under different concentrations of YE medium, the optimal concentration of induction medium can be determined; in addition to the above two methods, there are also induction methods such as cellophane and resin.

[0004] When the locust hind wings are used as a substrate for the induction of appressorium, the effect is often better in entomogenous fungi whose hosts are Orthoptera or Coleoptera insects. However, in the entomogenous fungi (Metarhizium brevis) whose hosts are Lepidoptera, especially Lepidoptera larvae, there are no reports of successful induction of appressorium on the hind wings of locusts. Similarly, the induction method with YE medium liquid, in addition to the poor effect and easy formation of mycelium, is also more complicated to explore the concentration. The use of cellophane, resin, etc. has more or less species specificity and poor effect. Therefore, the means of inducing appressorium still needs further exploration. Summary of the invention

[0005] The present invention provides a simple method for inducing appressorium of Metarhizium reinhardtii, which will provide theoretical and technical means for exploring genes related to appressorium formation, analyzing the infection mechanism of entomogenic fungi, and creating highly virulent Metarhizium reinhardtii strains, and can improve the efficiency of selecting highly virulent Metarhizium reinhardtii strains, thereby achieving efficient disease and insect control.

[0006] The present invention is achieved through the following technical solution: a simple method for inducing the attachment cells of Metarhizium anisopliae, comprising the following steps:

[0007] S1: Preparation of water agar: weigh 1.8 g of agar powder into a 250 mL volumetric flask, add 150 mL of pure water, mix well and sterilize at 121°C to obtain water agar. After sterilization, pour into a flat plate culture dish and cool for use;

[0008] S2: Preparation of qualitative filter paper: sterilize the qualitative filter paper at high temperature, cut it into 4 equal parts in a clean bench, and spread the filter paper pieces flat on the flat culture dish prepared in step S1;

[0009] S3: Treatment of coverslips: sterilize the square coverslips at high temperature and carefully place the coverslips on the surface of the filter paper in step S2;

[0010] S4: Preparation of spore suspension: The primary strain of Metarhizium anisopliae was transferred and cultured for 7-10 days until a large number of spores were produced, and 0.05% Tween 80 was used as the solvent to prepare 1x10 6 The conidia suspension with a concentration of pcs / mL is ready for use;

[0011] S5: Drop coating: Take 50 μL of the conidia suspension in step S4, drop it on the upper surface of the cover glass in the flat culture dish in step S3, and spread it over the entire glass slide to obtain an induction system of attached cells;

[0012] S6: Cultivation: Carefully seal the culture dish of the induction system in step S5 and place it in a 25°C light incubator for 5 days. Observe the formation of attachment cells under a microscope and analyze the induction results.

[0013] Furthermore, in the steps S1 and S2, the size of the flat culture dish and the shaped filter paper is both 90 mm.

[0014] Furthermore, in the step S1, the sterilization time is 15 minutes, and the concentration of water agar used is 1.0-1.2%;

[0015] Furthermore, in the step S3, the size of the square cover glass is 2 cm x 2 cm.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: a simple method for inducing the attachment cells of Metarhizium reinhardtii, which uses water agar as a substrate, qualitative filter paper as an intermediate layer of water-retaining medium, and a cover glass as a hydrophobic surface carrier, and drops conidia on a glass slide to induce the formation of attachment cells. The induction method of the present invention is simple and easy to operate, and has a good effect of inducing the formation of attachment cells, thereby enabling rapid screening of key genes for the formation of the infection structure of the attachment cells of Metarhizium reinhardtii, and also helping to create Metarhizium reinhardtii with excellent virulence. Therefore, the present invention provides an important experimental basis for the further industrial application of Metarhizium reinhardtii biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the embodiments and experimental examples of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments and experimental examples of the present invention. In the drawings:

[0018] Figure 1 This is a colony diagram of Metarhizium anisopliae of the present invention;

[0019] Figure 2 This is a diagram of a Lepidoptera larvae infected by Metarhizium brevis in the present invention;

[0020] Figure 3 This is a diagram of the construction process of the appressorium induction system of the present invention;

[0021] Figure 4 This is a comparison chart of the effects of the induction method of the present invention and the induction method of the prior art. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the embodiments and drawings. The following embodiments are only several specific embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any non-substantial changes to the present invention using this concept shall be deemed as an act of infringing the protection scope of the present invention.

[0023] Example 1

[0024] A simple method for inducing appressorium of Metarhizium anisopliae comprises the following steps:

[0025] S1: Preparation of water agar: weigh 1.8 g of agar powder into a 250 mL volumetric flask, add 150 mL of pure water, mix well and sterilize at 121°C for 15 min to obtain water agar with a concentration of 1.0-1.2%. After sterilization, pour into a 90 mm flat plate culture dish and cool for use;

[0026] S2: Preparation of qualitative filter paper: sterilize the 90 mm qualitative filter paper at high temperature, cut it into 4 equal parts in a clean bench, and spread the filter paper pieces flat on the 90 mm flat culture dish prepared in step S1;

[0027] S3: Treatment of coverslips: sterilize a 2 cm x 2 cm square coverslip at high temperature and carefully place the coverslip on the surface of the filter paper in step S2;

[0028] S4: Preparation of spore suspension: The primary strain of Metarhizium anisopliae was transferred and cultured for 7-10 days until a large number of spores were produced, and 0.05% Tween 80 was used as the solvent to prepare 1x10 6 The conidia suspension with a concentration of pcs / mL is ready for use;

[0029] S5: Drop coating: Take 50 μL of the conidia suspension in step S4, drop it on the upper surface of the cover glass in the flat culture dish in step S3, and spread it over the entire glass slide to obtain an induction system of attached cells;

[0030] S6: Cultivation: Carefully seal the culture dish of the induction system in step S5 and place it in a 25°C light incubator for 5 days. Observe the formation of attachment cells under a microscope and analyze the induction results.

[0031] The colony of Metarhizium anisopliae of the present invention is shown in FIG. Figure 1 As shown; the construction of the appressorium induction system of the present invention includes steps S1-S3, and the entire construction process is as follows Figure 3 As shown; the Metarhizium anisopliae used in the present invention is used to infect Lepidoptera larvae, such as Figure 2 The present invention also selected two prior art methods for inducing the formation of appressorium for comparison, namely, using cellophane as a medium to induce appressorium and using YE culture medium to induce appressorium. The two methods are prior art methods, and the present invention will not elaborate on their specific methods. The prior art induction method and the induction method of the present invention are compared. After 5 days of induction, as shown in FIG. Figure 4 As shown, it can be seen that the induction method of the present invention has good effect and is superior to the existing induction method. In addition, the method of the present invention is simple and therefore has outstanding advantages.

[0032] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A simple method for inducing appressorium of Metarhizium anisopliae, characterized in that: The steps include: S1: Preparation of water agar: weigh 1.8 g of agar powder into a 250 mL volumetric flask, add 150 mL of pure water, mix well and sterilize at 121°C to obtain water agar. After sterilization, pour into a flat plate culture dish and cool for use; S2: Preparation of qualitative filter paper: sterilize the qualitative filter paper at high temperature, cut it into 4 equal parts in a clean bench, and spread the filter paper pieces flat on the flat culture dish prepared in step S1; S3: Treatment of coverslips: sterilize the square coverslips at high temperature and carefully place the coverslips on the surface of the filter paper in step S2; S4: Preparation of spore suspension: The primary strain of Metarhizium anisopliae was transferred and cultured for 7-10 days until a large number of spores were produced, and 0.05% Tween 80 was used as the solvent to prepare 1x10 6 The conidia suspension with a concentration of pcs / mL is ready for use; S5: Drop coating: Take 50 μL of the conidia suspension in step S4, drop it on the upper surface of the cover glass in the flat culture dish in step S3, and spread it over the entire glass slide to obtain an induction system of attached cells; S6: Cultivation: Carefully seal the culture dish of the induction system in step S5 and place it in a 25°C light incubator for 5 days, then observe the formation of attachment cells under a microscope and analyze the induction results; In the steps S1 and S2, the size of the flat culture dish and the qualitative filter paper are both 90 mm; In the step S1, the sterilization time is 15 minutes, and the concentration of water agar used is 1.0-1.2%; In the step S3, the size of the square cover glass is 2 cm x 2 cm.

Images