GB/T 31792-2015 in English

Detailed explanation of detection method

Step 1: Symptom inspection and sample collection

• Collect the stems, diseased leaves or sunflower discs of suspected diseased plants, and pay special attention to the junction between diseased and healthy plants.
• Pick out shriveled, discolored, shrunken seeds and plant residues.

Step 2: Isolation and culture

• After surface disinfection of the diseased plant samples, inoculate them in SPDA medium containing streptomycin.
• Seed samples need to remove the seed coating agent and undergo surface sterilization before inoculation.

Step 3: Biological identification

• Observe the culture characteristics and morphological characteristics of the pathogen, and record the characteristics of conidia and spore apparatus.
• Observe the typical structure of α-type and β-type conidia under a microscope.

Step 4: Molecular biology detection

• DNA extraction: The pathogen DNA is extracted using the CTAB method to ensure purity and concentration.
• Real-time fluorescence PCR detection: Use specific primers DH-F, DH-R and probe DH-Pr for amplification, and the Ct value determines the positive result.
• ITS gene sequencing: Amplify the ITS gene fragment by PCR and confirm the homology with the GenBank sequence.

Standard background and technological evolution

This standard was formulated based on the needs of plant quarantine, combining traditional morphological identification and modern molecular biology technology. Real-time fluorescence PCR and ITS gene sequencing ensure the accuracy and sensitivity of detection.