GB/T 33117-2026Detection and identification of Oculimacula yallundae (English PDF)

9.2 Isolation and Culture

9.2.1 Seed Samples Weigh 50g of seeds to be tested, place them in a 250mL sterile Erlenmeyer flask, add 100mL of distilled water and 1-2 drops of polysorbate 20. (Tween 20), after sealing with aluminum foil, place in a reciprocating shaker and wash at low speed for 5 minutes; filter the washing suspension through clean gauze and then inject... Transfer the solution to a graduated centrifuge tube and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, combine all the washing suspensions, and centrifuge again to collect the precipitate. Transfer the sediment to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, remove the supernatant, air dry the precipitate at room temperature, and then dilute it 10-fold with sterile water. Spread the culture onto selective medium plates and incubate at 20°C for 7-14 days, then observe the colony growth.

9.2.2 Samples of diseased or damaged parts Suspected discolored tissues screened by symptom examination were cut into 3mm-5mm segments, soaked in 70% (volume fraction) ethanol for 30 seconds, and then... Disinfect with 1% sodium hypochlorite solution for 5 minutes, rinse 3 times with sterile water, blot dry with sterile filter paper, and then inoculate onto PDA plates; place the plates at 20°C. Incubate in an incubator for 7-14 days and observe the colony growth.

9.2.3 Soil Samples Weigh 2g of soil and add it to.200mL of sterile water. Stir with a magnetic rod for 30 minutes to mix thoroughly. Dilute the suspension 10 times and take 1mL to spread on a plate. Plates were placed on selective culture media and incubated at 20°C for 7-14 days, during which colony growth was observed.

9.2.4 Induction of conidia For the mycelia obtained in 9.2.1, 9.2.2, and 9.2.3, sporulation was induced using water agar medium (WA, prepared in accordance with Appendix B). Two-week-old mycelia were inoculated onto WA medium and cultured at 9°C for 7-9 days to produce conidia; mycelia were then inoculated onto PDA or spore suspensions. The conidia were coated onto the surface of a PDA and incubated at 18°C~21°C for 4 days to obtain more conidia. The induced conidia were then rinsed with sterile water and... After appropriate dilution, the strains were prepared for observation (microscopic morphology) and purification.

9.3 Molecular biological detection

9.3.1 DNA Extraction This method is applicable to mycelia isolated from seeds, diseased plant debris, soil samples, and from sections 9.2.1, 9.2.2, and 9.2.3.The extraction method can utilize hexadecyl sulfate. The CTAB method, commercial kits, or fully automated nucleic acid extractors are used. The specific operating procedures are detailed in Appendix B.

9.3.2 PCR detection Using the extracted total DNA as a template, PCR amplification was performed using primers specific to wheat basal rot. The specific operating steps are as described in Appendix B.

10.2 Morphological characteristics of pathogens

10.2.1 Hyphae Characteristics The fungal hyphae exist in two types. yellowish-brown linear branching vegetative hyphae, and dark, thick-walled stromata composed of swollen polygonal cells. Mycelial clusters.

10.2.2 Asexual characteristics Conidiophores are oppositely branched, transparent, and have 0 to 3 septa; conidiophores are subcylindrical to inverted flask-shaped, measuring (9 µm~ (23µm)×(2.5µm~5µm); conidia are transparent and smooth, slender and straight or slightly curved, with a sharp apex and a truncate base, containing 3 to 7 septa. Membrane, size (21µm~58µm)×(1.5µm~2µm) (see Appendix C).

10.2.3 Exhibits sexual characteristics (uncommon) The apothecia are

0.5 mm to

1.5 mm in diameter, densely clustered without stalks; the asci are (37 µm to 60 µm) × (4 µm to 6 µm), each containing 8 apothecia. Ascospores; ascospores (7µm~11µm)×(1.5µm~2µm), transparent, without septa or rarely with 1 septum (see Appendix C).

11 Result Determination The test results can be determined according to the following procedure.

a) Priority Process. Molecular biological detection and morphological characteristic verification, applicable to various samples such as seeds, diseased plant debris, and soil. If molecular... A negative biological test indicates that the wheat basal rot pathogen was not detected; if the test is positive, further isolation and culture of the pathogen is required, and the isolated material... If the asexual/sexual characteristics meet the identification characteristics in 10.2, it is considered detected; otherwise, it is not detected.

b) Alternative procedure. Isolation and culture molecular biological detection. If no suspicious colonies are found after isolation and culture according to 9.2, it is considered undetectable. The pathogen causing wheat basal rot was detected; if suspicious colonies or strains were found, DNA extraction and PCR testing should be performed according to 9.3, and positive results should be detected. A positive result indicates detection, a negative result indicates no detection; or continue culturing the suspected colonies, if their culture characteristics meet the identification criteria in Chapter 10. If the symptom is present, it can be directly determined as detected; otherwise, it is not detected.

12 Sample Preservation and Processing Samples should be properly stored after registration and signature by the person in charge, and a sample label should be affixed. Positive samples detecting wheat basal rot should be stored for at least [duration missing]. The samples should be stored for 6 months. After the storage period expires, they should be sterilized at high temperature.

13.Preservation and processing of bacterial strains Strains can be preserved for short or long periods. mycelial blocks can be placed in sterile water and stored at 4°C for short periods; regular preservation should be performed annually. To preserve the mycelium, suspend the mycelial blocks in 30% glycerol and store them in an ultra-low temperature freezer at -70°C for long-term preservation. Other suitable methods can also be used for preservation. Storage. Strains that do not require long-term storage should be autoclaved promptly.

14 Results Recording and Data Preservation The laboratory should maintain complete experimental records and related result images. GB/T 33117-2026. Quarantine Identification Method for Wheat Basal Rot Pathogen ICS

16 National Standards of the People's Republic of China Replaces GB/T 33117-2016 Quarantine identification method for wheat basal rot pathogens Published on 2026-03-

31 Implemented on October 1, 2026 State Administration for Market Regulation The State Administration for Standardization issued a statement.