ITS-RFLP method for rapidly identifying main cotton pathogenic fungus

A technology of pathogenic fungi and identification method, applied in the field of plant pathology, to achieve the effect of simple reaction procedure, low false positive rate and accurate identification

Inactive Publication Date: 2014-04-02
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It takes about a week to complete a series of ITS cloning, sequencing, and comparison procedures, and the identification cost of a sample is about 40 yuan

Method used

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  • ITS-RFLP method for rapidly identifying main cotton pathogenic fungus
  • ITS-RFLP method for rapidly identifying main cotton pathogenic fungus
  • ITS-RFLP method for rapidly identifying main cotton pathogenic fungus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The extraction of embodiment 1 cotton pathogenic fungus genomic DNA

[0033] 1) Scrape 50-100 mg of fresh mycelium from the PDA solid medium, put it in a 1.5 ml centrifuge tube, add 200 μl of preheated DNA extraction buffer [recipe: 50 mmol / L Hris-HCl (pH=7.5), 50mmol / L EDTA (pH=8.0), 1.4mol / L Nacl, 20g / L CTAB], grind it with a micro-grinding rod, then add 450μl extraction buffer, bathe in 65°C water for 30min, reverse every 10min;

[0034] 2) Add an equal volume of 1:1 saturated phenol and chloroform, mix well by inverting up and down, and centrifuge at 12000g for 15min at room temperature;

[0035] 3) Carefully pipette the supernatant into a new 1.5ml centrifuge tube, add an equal volume of ice-cold absolute ethanol to precipitate, place at room temperature for 10min, centrifuge at 12000g for 10min to collect the precipitate, discard the supernatant, and dry in an oven at 37°C;

[0036] 4) Add 30μl of ddH 2Dissolve genomic DNA in O and store in -20°C refrigerator fo...

Embodiment 2

[0038] Embodiment 2 Cotton pathogenic fungus ITS amplification

[0039] 1) Amplification reaction system: using the genomic DNA of the above-mentioned pathogenic fungi as a template, using the primer pair ITS4 5'-TCC TCC GCT TAT TGA TAT GC-3' and ITS5 5'-GGA AGT AAA AGT CGT AAC AAG G-3' Perform PCR amplification. 20μl PCR reaction system: including 10×PCR buffer (Mg 2+ ) 2 μl, 1.6 μl of dNTP solution with a concentration of 10 mM, 1 μl of each primer ITS4 / ITS5 with a concentration of 10 uM, 1 μl of genomic DNA, 0.2 μl of Taq enzyme with a concentration of 5 U / μl, and 13.2 μl of ddH 2 O.

[0040] 2) The amplification reaction program is: pre-denaturation at 94°C for 2 minutes, followed by denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 72°C for 40s, a total of 25 cycles, and finally extension at 72°C for 10 minutes to obtain the PCR product of the internal transcriptional spacer .

[0041] 3) For electrophoresis detection, take 5 μl of ITS PCR product,...

Embodiment 3

[0042] The selection of embodiment 3 restriction endonucleases

[0043] Screen for suitable four-base restriction enzymes. Based on the ITS sequence determination results of six cotton pathogenic fungi, the recognition of nine four-base restriction endonucleases (Acc II, AfaI, AluI, HaeIII, HhaI, Sau3A, MspI, TaqI and XspI) on different ITSs were analyzed one by one site, calculate the length of the product, fully consider the characteristics of polymorphism and fungal species specificity, and finally select four common endonucleases HhaI, HaeIII, TaqI, Sau3A.

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Abstract

The invention discloses an ITS-RFLP method for rapidly identifying main cotton pathogenic fungus, which mainly comprises the following steps: 1) extracting cotton main pathogenic fungus mycelia genome DNA, which comprises Verticillium dahliae, Verticillium alboatrum, Rhizoctonia solani, Fusarium moniliforme, Trichothecium roseum and Fusarium oxysporum; 2) amplifying ribosome internal transcribed spacer (ITC) using PCR: carrying out rapid amplification using general primer ITS4 and ITS5 of fungi ITS; 3) carrying out enzyme digestion reaction using restriction enzymes and identifying band spectrums: carrying out single digestion reaction of ITS products of cotton main pathogenic fungus randomly using restriction enzymes HhaI, HaeIII, TaqI and Sau3A whose recognition sites are four basic groups, and comparing ITS-RFLP band spectrum correlation tables and standard electronic maps, thereby rapidly identifying classes of pathogens. Compared with traditional time-consuming and labor-consuming morphological and physiological identification as well as ITS clone sequencing identification method of pathogen with high cost, the invention has the advantages of rapid, accuracy and economy, and simultaneous identifications of a plurality of pathogenic fungus.

Description

technical field [0001] The invention relates to the field of plant pathology, in particular, the invention relates to a rapid identification method of ITS-RFLP, the main pathogenic fungus of cotton. Background technique [0002] Cotton is an important economic crop and textile industry raw material worldwide, and it is also an important material related to the national economy and people's livelihood. For a long time, the widespread occurrence of cotton diseases and insect pests has always been the bottleneck problem restricting the high yield of cotton. Among them, Verticillium wilt and Fusarium wilt are the most serious, followed by boll disease and seedling disease, and most of them are fungal diseases. In order to formulate more scientific and reasonable prevention and control measures, the identification of disease types is the most necessary prerequisite, so it is particularly important to quickly and accurately identify pathogenic bacteria. [0003] The traditional i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/77C12R1/645
CPCC12Q1/04C12Q1/6858C12Q2535/138
Inventor 李志芳朱荷琴冯自力赵丽红师勇强
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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