Identification method for determining anastomosis groups of rhizoctonia solani

A technology of a rice sheath blight fungus and an identification method, which is applied in the identification field of the determination of the fusion group of the rice sheath blight fungus, and can solve the problems of inability to distinguish multiple fusion group types, disadvantage, insufficient rice sheath blight fungus, etc.

Inactive Publication Date: 2010-08-04
北京达成生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, rice sheath blight is a collection of species, and the existing identification methods for rice sheath blight are far from enough, and new technologies are still needed to further supplement and improve
The traditional mycelial fusion judgment standard has many main factors, which is not conducive to the objective and accurate judgment of the fusion group type of rice sheath blight
Current molecular techniques can only determine a few fusion group types, but cannot distinguish multiple fusion group types

Method used

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  • Identification method for determining anastomosis groups of rhizoctonia solani
  • Identification method for determining anastomosis groups of rhizoctonia solani
  • Identification method for determining anastomosis groups of rhizoctonia solani

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Isolation and purification of rice sheath blight from susceptible rice stalks

[0044] The rice stems of Guodao No. 6 hybrid rice susceptible to sheath blight were collected from the rice fields of the Fuyang Experimental Base of the China Rice Research Institute and brought back to the laboratory. Rinse the diseased stem tissue three times with sterile water, and blot the moisture on the surface of the tissue with sterilized filter paper. Then cut the diseased tissue into small pieces with a length and width of 5 mm, place it in 2% water agar (20 g of agar, 1000 mL of water) medium, and culture it in an incubator at 28 ° C. After 24 to 36 hours, silk grows around the diseased tissue. The colony with Sclerotinia characteristics was cut out with a sterile blade and transplanted into a PDA (200g potato, 20g glucose, 20g agar, 1000mL water) medium for purification. After purification and culture in an incubator at 28°C, it was stored in Store in the refrigerator...

Embodiment 2

[0045] Example 2 Extraction of bacterial strain genomic DNA

[0046] Acquisition of mycelium: After activation of the standard strain of Rhizoctonia solani or isolated rice sheath blight bacteria preserved on the slant medium, pick a small piece of mycelium (about 0.5mm×0.5mm) Transfer to a 250mL Erlenmeyer flask filled with 100mL PDB (200g potato, 20g glucose, 1000mL water) medium, and shake and culture at 28°C for 5-6d. The obtained culture was rinsed twice with sterilized water, then the culture was fully dried with a vacuum pump, and stored in a -20°C refrigerator for later use.

[0047] DNA extraction: The total genomic DNA of the strains was extracted by the improved CTAB method. Take 0.2 g of blotted hyphae, grind it with liquid nitrogen, add 800 μL DNA extraction solution (1L solution contains 20 g CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA and 1 ml β-mercaptoethanol, pH value is 8.0), in Incubate in a 1.5mL centrifuge tube at 65°C for 30min, shake gently 3-4 time...

Embodiment 3

[0051] Example 3 PCR Amplification of the ITS-5.8S rDNA Sequence of Different Mycelia Fusion Group Standard Strains

[0052] For the standard strains R1-R8 belonging to different hyphae fusion group strains AG-1IA, AG-1IB, AG-2-1, AG-4, AG-5, AG-6, AG-8 or AG-BI, carry out PCR amplification of ITS-5.8S rDNA sequence. Universal primers ITS1 and ITS4 designed by White et al. (1990) were synthesized by Shanghai Bioengineering Co., Ltd. The sequences of ITS1 and ITS4 are: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'; SEQ No.1), ITS4 (5' - TCCTCCGCTTATTGATATGC-3'; SEQ No. 2). The PCR reaction system is: 2.5 μL of 10×Taq reaction buffer in a total volume of 25 μL, 25 mM MgCl 2 , 2.5mMdNTP, 10μM primer ITS1, 10μM primer ITS4, standard strain DNA 100ng, 0.5U Taq DNApolymerase, add ddH 2 O to make up the volume to 25 μL. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 50 s, annealing at 55°C for 50 s, and extension at 72°C for 50 s, a total of ...

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Abstract

The invention relates to an identification method for determining anastomosis groups of rhizoctonia solani, which belongs to field of biotechnology. The identification method comprises the steps that: on the basis of processes of the extraction on total DNA of bacterial strains in eight different anastomosis groups of rhizoctonia solani, PCR amplification, the measurement and analysis of rDNA-ITS sequences and the like, basic group loci for identifying the bacterial strains in the different anastomosis groups of the rhizoctonia solani are found, high-specificity identification primers for identifying the bacterial strains in the different anastomosis groups are respectively designed, and the high-specificity identification primers are combined with a general primer ITS4 and a general primer ITS1 for detection by a one-step double PCR method, so that DNA fragments with specificity of 500 bp and 700 bp are obtained, and the identification of the high-specificity PCR of the bacterial strains in the different anastomosis groups is performed successfully. The identification method provides a quick and accurate molecular detection method for the identification of the rhizoctonia solani and provides theoretical reference for making the comprehensive treatment decision of multiple diseases such as banded sclerotial blight, rhizoctonosis and the like caused by formulating the rhizoctonia solani.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying fusion groups of rice sheath blight bacteria. Background technique [0002] "Food is the most important thing for the people, and rice is the first food." Rice is the main food crop in our country and plays an important role in our national economy and agricultural production. In recent years, due to the outbreak of various rice disease epidemic races and the variation of their pathogenicity, the degree of damage has increased, which seriously threatens the production safety of rice. Rice sheath blight is one of the worldwide diseases that occur widely in rice producing areas and cause serious damage. With the vigorous promotion of short-stalked and multi-tiller hybrid rice combinations, the increase in fertilizer application and planting density, and climate warming, the occurrence and damage of rice sheath blight has become increasingly seriou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 王玲黄世文刘连盟
Owner 北京达成生物科技有限公司
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