Method for separating rhizoctonia solani fusion group AG-3 basidiospore

Abstract

The invention discloses a rhizoctonia solani fusion group AG-3 basidiospore separation method, and belongs to the technical field of biology, the rhizoctonia solani fusion group AG-3 basidiospore separation method comprises the following steps: S1, preparing a rhizoctonia solani fusion group AG-3 spore suspension; and S2, collecting tender tobacco leaves, disinfecting, inoculating the spore suspension obtained in the step S1 on the disinfected tobacco leaves, culturing, regularly sampling and observing, and separating and purifying the scabs again after the scabs are observed, so as to obtain a bacterial strain which is a newly formed basidiospore bacterial strain after spore germination. The invention discloses a method for inducing rhizoctonia solani fusion group AG-3 basidiospore by using tobacco leaves, the whole separation method is simple to operate, and analysis and research on pathogenicity, pathogenic mechanism, polygene systematics and genetic diversity of a rhizoctonia solani fusion group AG-3 single spore strain are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a method for isolating basidiospores of Rhizoctonia solani fusion group AG-3. Background Technology

[0002] Tobacco target spot is a leaf spot disease caused by *Rhizoctonia solani*. The pathogen of tobacco target spot is *Rhizoctonia solani* subgroup AG-3-TB. The asexual generation of *Rhizoctonia solani* is (…). Rhizoctonia solani It belongs to the phylum Ascomycota, class Hyphomycetes, order Asporales, genus Rhizoctonia, and its sexual generation is *Citrus fulvidracoides*. Cucumber beetle It belongs to the phylum Basidiomycota, class Agaricomycetes, order Keratopterales, and genus Apocynum.

[0003] In terms of classification, the most widely accepted classification method for Rhizoctonia solani is based first on the number of cell nuclei, dividing them into uninucleate Rhizoctonia. Rhizoctonia spp., UNR), binucleate Rhizoctonia spp., BNR) and multinucleate rhizobium Rhizoctonia (spp, MNR); and then divided into different fusion groups according to the hyphal fusion characteristics. Among them, *Rhizoctonia solani* is the most well-known, and currently it is divided into 14 fusion groups, AG-1~AG-13 and AG-BI, based on hyphal fusion experiments. The fusion group system is considered the most effective classification method for *Rhizoctonia solani*, as different fusion groups can be distinguished according to the fusion behavior of *Rhizoctonia solani*, but it cannot effectively divide different subgroups within the same fusion group. The same fusion group can be divided into different fusion subgroups according to the homology of the 5.8S rDNA-ITS sequence and the differences in the host. Among them, there are at least 6 hyphal fusion groups that harm tobacco, namely AG-1~AG-6, but the infection sites and symptoms of different groups are somewhat different. AG-1, AG-4 and AG-5 mainly cause damping-off and stem rot in tobacco seedlings, while AG-2, AG-3 and AG-6 mainly cause tobacco leaf spot. In recent years, tobacco target spot disease has seriously affected the quality and grade of tobacco, and the production of sexual spores has further aggravated the occurrence and damage of the disease.

[0004] Current research on the pathogenic mechanism of AG-3, both domestically and internationally, mainly focuses on mycelial infection mechanisms (sclerotium formation, cell wall degrading enzymes, crude toxins, and genetic differentiation). However, because the infection mode of this pathogen differs from other Rhizoctonia solani species, the emergence of sexual spores provides a basis for systematically understanding the pathogenicity, pathogenic mechanisms, and genetic diversity changes of AG-3 sexual spore progeny, while also posing new challenges to the control of Rhizoctonia solani diseases. Therefore, inducing sexual spores of the tobacco target spot pathogen and conducting research on the pathogenicity, cell wall degrading enzymes, toxins, multi-gene phylogenetic analysis, and genetic diversity of the progeny monocellular strains of sexual spores can lay the foundation for further understanding the pathogenic mechanism and genetic differentiation of Rhizoctonia solani. The research results can reveal the role of sexual spores in pathogenicity differentiation and genetic diversity, and also provide the theoretical conditions for formulating control strategies and carrying out effective control of tobacco target spot disease.

[0005] Rhizoctonia solani AG-3 is generally asexual, and its sexual form is difficult to detect. During natural infection, Rhizoctonia solani mainly reproduces asexually as hyphae or sclerotia. Because the spores of Rhizoctonia solani are small and the induced sexual spores are often accompanied by a large number of other bacteria, isolation is often contaminated by these bacteria, and common single-spore isolation methods cannot isolate single-spore strains. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the present invention proposes a method for isolating basidiospores of Rhizoctonia solani fusion group AG-3.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for isolating basidiospores of Rhizoctonia solani fusion group AG-3 includes the following steps: S1, prepare Rhizoctonia solani fusion group AG-3 spore suspension; S2: After collecting and disinfecting young tobacco leaves, inoculate the spore suspension obtained in step S1 onto the disinfected tobacco leaves for culture. Sampling and observation are performed periodically. Once lesions are observed, samples are taken from the lesions to check for the presence of filamentous hyphae. If hyphae are present, a 1cm sample is taken from the lesions. 2 The lesions of different sizes were cut out with scissors and inoculated onto PDA agar plates. The isolated fungi were examined under a microscope, and the resulting Rhizoctonia solani hyphae were the basidiospore strains newly formed after spore germination.

[0008] Furthermore, the disinfection method for tobacco leaves in step S2 is as follows: the collected leaves are first treated with 1.5% sodium hypochlorite for 1 minute, then washed 2-3 times with sterile ddH2O, and the moisture on the leaf surface is absorbed with sterile filter paper.

[0009] Furthermore, in step S2, the culture conditions after the tobacco leaves are inoculated with the suspension are: 12 hours of light per day, cultured in a light incubator at 28°C and 80% humidity.

[0010] Further, in step S1, the method for preparing the Rhizoctonia solani fusion group AG-3 spore suspension is as follows: scrape the hymenium of the observed basidiospores with tweezers, place it in a 50 mL centrifuge tube, add an appropriate amount of sterile water to make a crude spore suspension, take an appropriate amount of the crude suspension, shake it, and filter it with lens paper in a clean bench. Examine the filtrate under a microscope to ensure that there are no mycelial residues in the filtrate and that the basidiospores can be observed. Dilute the filtrate with sterile water or increase the spore amount, and examine it under a hemocytometer until a spore concentration of 100 spores / mL is reached before use.

[0011] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The present invention discloses a method for inducing Rhizoctonia solani fusion group AG-3 basidiospores using detached tobacco leaves. The entire isolation method is simple to operate and is conducive to the pathogenicity, pathogenic mechanism, multigene systematics, and genetic diversity analysis of Rhizoctonia solani fusion group AG-3 single spore strain. Detailed Implementation

[0012] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0013] Example 1

[0014] The method for preparing the Rhizoctonia solani fusion group AG-3 spore suspension in this invention is as follows: 1. Under aseptic conditions, the preserved AG-3 strain (provided by Yunnan Agricultural University) was activated and purified 2-3 times for later use. Barley grains were moistened with deionized water and then dispensed into 100 mL glass culture bottles, 20 g of oat grains per bottle. The bottles were sealed and autoclaved (120℃, 30 min). After the barley grains cooled, under aseptic conditions, 6 mm diameter mycelial blocks of the strain, pre-activated on PDA medium, were added to the glass bottles containing the barley grains, 10 mycelial blocks per bottle. The bottles were incubated in the dark at 25℃ for 7 days until the grains were fully colonized.

[0015] 2. During periods of continuous rainy weather, first moisten the soil around the tomatoes that have been cultivated for 3 to 4 weeks. Disinfect the base of the tomato plant's stem (5 cm) with 75% alcohol, then use a sterilized toothpick to make a ring piercing of the disinfected area. Place 10 barley grains of colonizing mycelium as inoculum, 1 cm deep, in the soil around the base of the stem. After inoculation, cover the inoculum with plastic wrap to retain moisture. Maintain a temperature and humidity of over 80% and observe for signs of disease regularly.

[0016] 3. Approximately 14 days after inoculation, symptoms appear at the inoculation site of the plant, accompanied by the hymenium covering its surface. Samples are taken from the portion containing the fruiting body and prepared as 1×1 cm specimens for staining and observation. Staining is performed using safranin and KOH. First, stain with safranin for 1 minute, then rinse thoroughly with KOH. Tilt the slide at 45° and slowly rinse with KOH until no red liquid remains. Cover with a coverslip and observe under a microscope. First, locate the target under low magnification, then switch to high magnification to locate and observe the sporulation structure.

[0017] 4. Use tweezers to scrape the hymenium of the observed basidiospores and place it in a 50 mL centrifuge tube. Add an appropriate amount of sterile water to prepare a crude spore suspension. Take an appropriate amount of the crude suspension, shake it, and filter it with lens paper in a clean bench. Examine the filtrate under a microscope to ensure that there are no mycelial residues and that the basidiospores can be observed. Dilute the filtrate with sterile water or increase the spore count until a spore concentration of 100 spores / mL is reached. This yields the Rhizoctonia solani fusion group AG-3 spore suspension for later use.

[0018] Example 2

[0019] A method for isolating basidiospores of Rhizoctonia solani fusion group AG-3 includes the following steps: S1, take the AG-3 spore suspension of Rhizoctonia solani fusion group from step S1; S2: After collecting and disinfecting young tobacco leaves, inoculate the spore suspension obtained in step S1 onto the disinfected tobacco leaves for culture. Sampling and observation are performed periodically. Once lesions are observed, samples are taken from the lesions to check for the presence of filamentous hyphae. If hyphae are present, a 1cm sample is taken from the lesions. 2 The lesions of different sizes were cut out with scissors and inoculated onto PDA agar plates. The isolated fungi were examined under a microscope, and the resulting Rhizoctonia solani hyphae were the basidiospore strains newly formed after spore germination.

[0020] The disinfection method for tobacco leaves is as follows: the collected leaves are first treated with 1.5% sodium hypochlorite for 1 minute, then washed 2-3 times with sterile ddH2O, and the moisture on the leaf surface is absorbed with sterile filter paper.

[0021] The culture conditions for tobacco leaves after inoculation with the suspension were: 12 hours of light per day, cultured in a light incubator at 28°C and 80% humidity.

[0022] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for isolating basidiospores of Rhizoctonia solani fusion group AG-3, characterized in that: It includes the following steps: S1, prepare Rhizoctonia solani fusion group AG-3 spore suspension; S2: After collecting and disinfecting young tobacco leaves, inoculate the spore suspension obtained in step S1 onto the disinfected tobacco leaves for culture. Sampling and observation are performed periodically. Once lesions are observed, samples are taken from the lesions to check for the presence of filamentous hyphae. If hyphae are present, a 1cm sample is taken from the lesions. 2 The lesions of different sizes were cut out with scissors and inoculated onto PDA agar plates. The isolated fungi were examined under a microscope, and the resulting Rhizoctonia solani hyphae were the basidiospore strains newly formed after spore germination.

2. The method for isolating basidiospores of Rhizoctonia solani fusion group AG-3 according to claim 1, characterized in that: The disinfection method for tobacco leaves in step S2 is as follows: the collected leaves are first treated with 1.5% sodium hypochlorite for 1 minute, then washed 2-3 times with sterile ddH2O, and the moisture on the leaf surface is absorbed with sterile filter paper.

3. The method for isolating basidiospores of Rhizoctonia solani fusion group AG-3 according to claim 1, characterized in that: In step S2, the culture conditions after the tobacco leaves are inoculated with the suspension are: 12 hours of light per day, cultured in a light incubator at 28°C and 80% humidity.

4. The method for isolating basidiospores of Rhizoctonia solani fusion group AG-3 according to claim 1, characterized in that: In step S1, the method for preparing the Rhizoctonia solani fusion group AG-3 spore suspension is as follows: scrape the hymenium of the observed basidiospores with tweezers, place it in a 50 mL centrifuge tube, add an appropriate amount of sterile water to make a crude spore suspension, take an appropriate amount of the crude suspension, shake it, and filter it with lens paper in a clean bench. Examine the filtrate under a microscope to ensure that there are no mycelial residues in the filtrate and that basidiospores can be observed. Dilute the filtrate with sterile water or increase the spore amount, and examine it under a hemocytometer until a spore concentration of not less than 100 spores / mL is reached before use.