GB/T 18085-2026Detection and identification of Tilletia controversa J.G. Kühn (English PDF)

1.5

h) or moist heat (121°C, 20 min). Add 100mL of distilled water to the container, and then add 1-2 drops of the surfactant Tween-20 reagent.

9.3.1.2 Sealing Seal the Erlenmeyer flask with aluminum foil or sealing film.

9.3.1.3 Vibration Washing Place the Erlenmeyer flask on a reciprocating shaker and shake and wash for 5 minutes.

9.3.1.4 Centrifugation Immediately remove the Erlenmeyer flask and transfer the washing suspension into a 10mL-20mL graduated centrifuge tube that has been sterilized by dry heat. Centrifuge at 1000 rpm. Heart 3min.

9.3.1.5 Mixing and centrifugation Remove the centrifuge tube, discard the supernatant, add the remaining wash suspension, mix and centrifuge until all wash suspension has been centrifuged, and retain the sediment. sediment.

9.3.2 Fixed volume Add Schirr flotation agent to the precipitate to suspend it, and adjust the volume to 1mL~3mL depending on the amount of precipitate.

9.3.3 Production Use an adjustable micropipette to pipette 5 µL to 20 µL of the precipitate suspension onto a glass slide, then cover with a coverslip. (Precipitate suspension for slide preparation) The volume should be such that the suspension does not overflow after the cover glass is placed.

9.3.4 Microscopic examination For each test sample, the precipitate suspension should be examined on at least 5 slides, with each slide examined sequentially according to the field of view.

9.3.5 Measurement If suspected teliospores of wheat dwarf smut are found, randomly measure the ridge height of 30 mature teliospores. Measure 5 slides. If there are still fewer than 30 teliospores, increase the number of slides to be examined until all the precipitate suspension is used up.

9.3.1 Washing and centrifugation

9.3.1.1 Add water Pour the test sample into a 250 mL Erlenmeyer flask that has been sterilized by dry heat (165°C,

10.1.1 Or 10.1.2)

10.1.1 Microscopic identification method The morphology of teliospores is shown in Appendix A. Each teliospore was visualized by aligning it vertically and horizontally on the microscope monitor screen (or using an eyepiece micrometer). The height of four ridges was measured mechanically, and the average ridge height was calculated. This average ridge height was then used to represent the ridge height of the teliospore. Identification. If a gall is found, the average ridge height of 30 randomly measured mature teliospores should also be obtained.

10.1.2 Scanning Electron Microscopy Identification Method

10.1.2.1 Sample Preparation Perform the operation according to the sample preparation requirements of the selected scanning electron microscope.

10.1.2.2 Gluing the platform Use conductive double-sided tape to attach the dried sample to the sample stage.

10.1.2.3 Coating A metal film is sprayed onto the surface of the sample and sample stage to increase the sample's conductivity. Metals used for coating include gold, platinum, palladium, and silver. After coating, the product is observed.

10.1.2.4 Microscopic examination and measurement Follow the operating instructions for the scanning electron microscope instrument software. Examine at least five samples from each sample, ensuring all samples are examined within the field of view. Look. If suspected teliospores of wheat dwarf smut are found, randomly measure the height of the ridges of 10 to 30 mature teliospores and calculate the average ridge height. Ridge height value. If the number of teliospores is insufficient, increase the number of samples to be tested.

10.2 Autofluorescence Microscopic Identification Method for Teliospores

10.2.1 Production Scrape a small amount of teliospore powder from the gall onto a clean glass slide, add an appropriate amount of distilled water to prepare a teliospore suspension (concentration 10×100). (It is advisable to keep no more than 40 teliospores per field of view under magnification), and place them in a dust-free place to dry naturally. Then, collect the dried teliospores that have adhered to the slide. Add a drop of non-fluorescent microscope objective lens oil to the lens, cover with a coverslip, and then add the lens oil mentioned above.

10.2.2 Selecting the Filter Set The prepared slide was placed on an epifluorescence universal microscope with an excitation filter of 485 nm and a barrier filter of 520 nm.

10.2.3 Timing and Counting Determine the observation field and start timing simultaneously. After irradiating each field for

2.5 minutes, begin examining for positive and negative autofluorescence reactions. The required number of teliospores. The entire process should not exceed 3 minutes.

10.2.4 Calculate the percentage of autofluorescence At least 5 fields of view and.200 teliospores were observed in each gall sample. The percentage of teliospores showing positive autofluorescence was then calculated. This percentage represents the autofluorescence rate of the gall for identification.

10.3 Physiological Identification Methods for Teliospore Germination

10.3.1 Making a flat plate After being sterilized by conventional autoclaving and cooled to about 50°C, 3% water agar was poured into 9cm diameter petri dishes, 20mL per dish. When making flat plates, it is necessary to prevent the formation of surface water flow.

10.3.2 Coating Plate Take a portion of the gall and add an appropriate amount of sterile distilled water to prepare a teliospore suspension, which is then evenly spread onto a water agar plate. The concentration of the teliospore suspension... The optimal magnification is 40 to 60 teliospores per field of view at low magnification (10×10).

10.3.3 Cultivation Plates coated with teliospores were placed under continuous (or alternating with 12-hour darkness) light conditions at 5°C±1°C and 17°C±1°C. nourish.

10.3.4 Observation and Observation Time Observe the germination of teliospores under a microscope (10×10). The first observation can be taken after 10 days of culture, and thereafter every 3 to 7 days. Observe the germination of teliospores once and calculate the germination rate.

11.2 Discovery of fungal galls 11.2.1 11.2.2~

11.2.4 can all be used as independent identification methods, and a judgment can be made if any one of them is met.

11.2.2 The morphological characteristics of teliospores are as follows:

---When the average height of the reticulate ridges of 30 mature teliospores of a fungal gall is greater than or equal to 1.43 µm, it is considered a detection of wheat dwarf blackhead. germs;

---When the average height of the reticulate ridges of 30 mature teliospores of a gall is less than or equal to 0.70 µm, it is considered as not detecting wheat dwarf black fungus. Ear disease pathogen.

11.2.3 Microscopic characteristics of autofluorescence rate of teliospores (when the average height of the reticulate ridges of 30 mature teliospores of the gall is 0.71 µm~) (Performed at 1.42µm) as follows:

---When the autofluorescence rate of the gall teliospores is greater than or equal to 80%, it is considered that wheat dwarf smut fungus has been detected;

---When the autofluorescence rate of the gall teliospores is less than or equal to 30%, it is determined that the wheat dwarf smut fungus has not been detected;

---The autofluorescence rate of teliospores of fungal galls is 31%~79%, which needs to be determined in conjunction with the germination status.

11.2.4 The physiological characteristics of teliospore germination are as follows:

--- Germination at 5°C and no germination at 17°C indicates that wheat dwarf smut pathogen has been detected;

--- Germination can occur at both 5°C and 17°C, indicating that wheat dwarf smut fungus was not detected.