Method for detecting activity of rice kernel smut

Abstract

The invention discloses a method for detecting the activity of rice kernel smut. According to the method, propidium iodide is used as a coloring agent to carry out fluorescent staining on the rice kernel smut, and then the activity of the rice kernel smut is detected. The activity of the teliospore can be detected, only about 40 minutes are needed from sample preparation to detection completion, and the method can be used for detecting the activity of the pathogen at a port.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and particularly relates to a method for detecting the activity of rice smut fungus. Background Art

[0002] Rice grain smut is also called black powder grain or black rice grain. It is caused by the smut fungus (Tilletia horrida Tak.). A soil-borne fungal disease first described by Takahashi in 1896. It only harms rice grains and is common in our rice-producing areas. It occurs, and is more common in mid- and late-season rice. It is more serious in hybrid rice seed fields. The diseased grain rate is generally 10% to 30%. In serious cases, the rate can even reach 80%, affecting both hybrid rice seed production and seed quality.

[0003] Currently, the main detection method for rice grain smut is morphological identification, which involves observing and identifying its teliospores and sterile cells.

[0004] It is impossible to determine the activity of spores. Although the traditional spore germination method can detect spore activity, it requires sterile operation and is time-consuming. The length of the test is long, making it difficult to meet the rapid testing requirements of laboratories and ports. When powdery mildew spores are detected, quickly determining whether they are active is an important means to ensure customs clearance. Rapid evaluation of the spore inactivation effect after treatment is also an important technical means to ensure rapid customs clearance.

[0005] Since the 1960s, researchers have used fluorescent staining to identify cell activity. (Propidium Iodide, PI) can penetrate the cell membrane of dying or dead cells and bind to DNA / RNA The double-stranded base pairs are embedded and combined to produce red fluorescence, so dead cells or apoptotic cells can be detected by fluorescence. In the inhibition test of Trichothecium roseum using citric acid, the cell membrane was dyed with PI. Judge the completeness.

[0006] Acridine Orange (AO) can bind to DNA or RNA molecules through electrostatic attraction. AO is widely used in the detection of cell apoptosis by stimulating green fluorescence. The color technology is used to stain peripheral blood cells as a rapid detection of abnormal proliferation of hematopoietic tissue (such as bone marrow) (such as leukemia, myelodysplastic syndrome, etc.) Summary of the Invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provides a method for detecting the activity of rice smut fungus.

[0008] In order to achieve the above object, the technical solution provided by the present invention is: The method for detecting the activity of the rice smut fungus comprises the following steps: using propidium iodide as a dye to perform fluorescent staining on the rice smut fungus and then detecting the activity thereof.

[0009] Preferably, the initial concentration of propidium iodide in the propidium iodide solution during staining is 0.1 mg / mL-10 mg / mL, and the staining time is 10-30 min.

[0010] More preferably, the initial concentration of propidium iodide in the propidium iodide solution during staining is 0.1 mg / mL, and the staining time is 10 min.

[0011] Preferably, the activity detection is performed on the teliospores of Ustilago oryzae.

[0012] The above-mentioned method for detecting the activity of rice smut fungus specifically comprises the following steps: (1) Preparing a spore suspension of Ustilago oryzae; (2) Add propidium iodide stain solution to the spore suspension and stain in the dark; (3) The dyed spores are broken by external force to allow the spore contents to flow out; (4) Observe the contents of the spores under a fluorescence microscope: if the contents emit red fluorescence, they are judged to be inactive spores; if the contents do not emit red fluorescence, they are judged to be active spores.

[0013] Preferably, the volume ratio of the propidium iodide stain solution to the spore suspension is 1:199.

[0014] The present invention will be further described below: This study used the rice smut fungus Tilletia horrida Tak. as a test material and compared the staining effects of six fluorescent dyes at three different concentrations and three different staining time combinations. When the spores were intact, none of the six fluorescent dyes could clearly distinguish between live spores and inactivated spores treated at 121°C for 30 minutes. However, after externally damaging the stained spores to release their contents, propidium iodide-stained dead spores emitted red fluorescence, while live spores did not, clearly distinguishing between live and dead spores. This study has established a preliminary propidium iodide (PI)-based fluorescence staining method for detecting the activity of Tilletia horrida Tak., a rice smut fungus. This method can detect the activity of teliospores, taking only approximately 40 minutes from sample preparation to detection. It is suitable for port activity testing of this pathogen.

[0015] In summary, the present invention selected six fluorescent dyes, including propidium iodide and acridine orange, and combined them with fluorescence microscopy to carry out activity detection research on rice smut fungus. Ultimately, propidium iodide was determined to be the most suitable dye, and a rapid, accurate, and reproducible method for detecting spore activity was established, providing a scientific basis for risk assessment of rice smut and evaluation of pest control treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Optical microscope observation of teliospores and sterile cells of the test materials (×400); Figure 2 :The effect of propidium iodide on the staining of intact spores of Ustilago oryzae; Note: A and B are the spore staining results of the control group, A is dark field, B is bright field, C and D are the spore staining results of the experimental group, C is dark field, D is bright field; Figure 3 : Spore contents of propidium iodide staining test group; Figure 4 : Spore contents of the control group stained with propidium iodide. DETAILED DESCRIPTION

[0017] 1 Materials and Methods 1.1 Test strains It was isolated from hybrid rice grown in the fields of Changsha County, Hunan Province that year.

[0018] 1.2 Reagents 1.2.1 Fluorescent dyes Six fluorescent dyes were used to detect the activity of pathogen spores, see Table 1.

[0019] Table 1 Fluorescent dyes tested Dye name Excitation wavelength (nm) Emission wavelength (nm) Fluorescent colors source Propidium iodide (PI) 534 617 red Solarbio Fluorescein diacetate (FDA) 488 530 green Solarbio Acridine orange (AO) 488 530 green Solarbio Calcein 488 530 green Solarbio DAPI 340 488 blue Solarbio SYTO-9 488 530 green Solarbio 1.2.2 Other chemical reagents Dimethyl sulfoxide (DMSO, Sinopharm Chemical Reagent Co., Ltd.); Acetone (Acetone, Sinopharm Chemical Reagent Co., Ltd.).

[0020] 1.3 Main instruments and equipment ZEISS upright fluorescence microscope (Axio Scope 5, ZEISS, Germany); autoclave (HICLAVE HV-85, HIAYAMA, Japan); centrifuge (3K15, SIGMA).

[0021] 1.4 Experimental Methods 1.4.1 Isolation and identification Weigh 50 g of freshly collected rice seeds suspected of harboring Ustilago oryzae and place them into a 250 mL sterile Erlenmeyer flask. Add 100 mL of sterile water and 2 drops of Tween-20. Seal the flask with aluminum foil and wash on a shaker for 5 minutes. Pour the wash suspension into a 10-20 mL sterile centrifuge tube and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, add the remaining wash suspension, and repeat the centrifugation until all the wash suspension has been centrifuged, retaining the precipitate. Resuspend the precipitate in sterile water according to the volume. Aspirate 10 μL of the suspension to prepare a slide and observe its morphology and structure under a microscope for identification.

[0022] 1.4.2 Fluorescent dye initial screening 1.4.2.1 Preparation of spore suspension Take the rice grains infected with rice smut, soak them in 75% ethanol for 1 min and then in 5% sodium hypochlorite for 10 min, then wash them three times with sterile water, put them on sterile filter paper to dry, and then peel them with a blade. Transfer the pathogen spores to a culture dish filled with sterile water to make 10 5 ~10 6 spores / mL of spore suspension.

[0023] 1.4.2.2 Fluorescent staining Prepare 4-5 mL of fresh spore suspension and transfer 199 μL of each treatment into a 1.5 mL centrifuge tube. Divide the suspension into two groups: one treated at 121°C for 30 min to produce dead spores (the experimental group), and one untreated group to produce live spores (the control group). Stain the suspension with each of the six dyes, prepare slides, observe under a fluorescence microscope, and save the images. Select a fluorescent dye that clearly distinguishes live from dead spores. Perform a single-factor orthogonal screening experiment with each dye at at least three different concentrations and three different treatment times, for a total of nine groups.

[0024] Table 2 Initial conditions and usage of dyes 1.4.3 Staining method Add 1 μL of dye to 199 μL of spore suspension, vortex to mix, and stain at room temperature in the dark. Immediately after the timer expires, centrifuge at 14,000 rpm for 1 minute and discard the supernatant. Wash with 200 μL of sterile water and centrifuge at 14,000 rpm for 1 minute. Repeat twice, then resuspend in 20 μL of sterile water. Prepare the suspension fresh for each fluorescent staining observation.

[0025] 1.4.4 Observation of dyeing effect Pipette 10 μL of the fluorescent dye-treated sample slide and place it on the stage of an upright fluorescence microscope. Locate the spores under low magnification, then switch to high magnification. Fine-tune the microscope to ensure a clear field of view. Observe the spore staining at the corresponding excitation and emission wavelengths. Also, select the optimal dye concentration and staining time based on the staining (staining rate and brightness). For live and dead spores, observe spores with weak fluorescence after fluorescent staining by gently pressing the slide to break the spore wall, allowing the spore contents to flow out and observe the staining of the spore contents.

[0026] 1.4.5 Germination rate experiment Spore suspension preparation: Place spore suspensions from the experimental and control groups in a lighted incubator for germination rate testing. Germination conditions: 28°C constant temperature, culture in a Petri dish, suspension thickness ≤ 1 mm, 70% light intensity, 12 hours of light per day. Observe germination starting after 7 days and continuing for 21 days. The experiment was repeated at least three times. Germination was defined as the presence of primary filaments from thick-walled spores. Pale yellow chlamydospores were considered immature and unable to germinate and were not counted. At least 200 spores were examined for each group.

[0027] 2 Results and Analysis 2.1 Isolation and identification The prepared spore suspension slides were observed under a microscope. The results showed that mature thick-walled spores were dark brown to black, opaque, and densely covered with warts on the outer wall; sterile cells were spherical, nearly spherical, transparent, and had smooth spore walls ( Figure 1 The morphological characteristics of the chlamydospores are consistent with those of the chlamydospores of Ustilago oryzae.

[0028] 2.2 Fluorescence staining results From the results of propidium iodide staining of the control group spores (A, B) and the experimental group spores (C, D) of Ustilago oryzae at a concentration of 0.1 mg / mL for 10 min, it can be seen that ( Figure 2 ), A and C can emit red fluorescence, and can also emit light under other treatment conditions. After the experimental group spores were crushed ( Figure 3), it was observed that the red fluorescent contents inside the spores flowed out after rupture. After continuous observation of 400 spores, all dead spores would flow out red contents after rupture. Figure 4 ), it was observed that, in addition to the spores with red fluorescent contents flowing out, there were also some spores whose contents did not emit red fluorescence (e.g. Figure 4 The red arrows indicate that PI did not enter the spores and the spores were active. After continuous observation of 400 spores, the proportion of active spores was about 32.3%. Figure 4 It can be seen that propidium iodide can distinguish live from dead spores at a concentration of 0.1 mg / mL and staining for 10 minutes, and the same can be said for other treatment conditions. The other five dyes were unable to penetrate the spores or distinguish between live and dead spores under nine different conditions, including three concentrations and three staining times (see Table 3).

[0029] Table 3 Dyeing results of 5 dyes Note: “+” indicates fluorescent color observed under a microscope, “-” indicates no fluorescent color observed under a microscope 2.3 Germination rate results The experiment showed that the spore germination rates of the control group were 28.8%, 26.4% and 30.5%, with an average germination rate of 28.6%; the winter spore germination rates of the experimental group were 0%, 0% and 0%, with an average germination rate of 0%.

[0030] 3 Results and Discussion Screening revealed that staining with propidium iodide, followed by external force to break the spores and observe the contents, clearly distinguished live and dead spores. The optimal staining conditions were 0.1 mg / mL, dark-protected, for 10 minutes. This study screened six fluorescent dyes for detecting spore viability in Ustilago oryzae, identifying an ideal fluorescent dye.

[0031] The viable spore rate obtained by PI staining was similar to the actual germination rate, further demonstrating that PI staining is feasible for detecting spore viability in rice grain smut. The staining results showed that the viable staining rate was slightly higher than the spore germination rate, suggesting that a small number of teliospores are in a passive state. Although they are viable, they are unable to germinate normally. This is consistent with Liu Zhanshan's experimental results on germination of rice grain smut. The dead spore staining rate and germination rate of the experimental group corroborated each other, indicating that the spores heat-treated at 121°C were completely inactivated and unable to germinate.

[0032] In summary, this experiment established a rapid, accurate and reproducible method for detecting spore activity, which provides a scientific basis for risk assessment of rice smut and evaluation of pest control effects.

Claims

1. A method for detecting the activity of rice smut fungus, characterized in that: The method comprises the following steps: using propidium iodide as a dye to perform fluorescent staining on the rice smut fungus and then detecting the activity of the fungus.

2. The method for detecting the activity of rice smut fungus according to claim 1, wherein: During staining, the initial concentration of propidium iodide in the propidium iodide solution is 0.1 mg / mL-10 mg / mL, and the staining time is 10-30 minutes.

3. The method for detecting the activity of rice smut fungus according to claim 2, wherein: During staining, the initial concentration of propidium iodide in the propidium iodide solution was 0.1 mg / mL, and the staining time was 10 min.

4. The method for detecting the activity of rice smut fungus according to any one of claims 1 to 3, wherein: The activity detection is to perform activity detection on the teliospores of Ustilago oryzae.

5. The method for detecting the activity of rice smut fungus according to claim 4, wherein: The method specifically comprises the following steps: (1) Preparing a spore suspension of Ustilago oryzae; (2) Add propidium iodide stain solution to the spore suspension and stain in the dark; (3) The dyed spores are broken by external force to allow the spore contents to flow out; (4) Observe the contents of the spores under a fluorescence microscope: if the contents emit red fluorescence, they are judged to be inactive spores; if the contents do not emit red fluorescence, they are judged to be active spores.

6. The method for detecting the activity of rice smut fungus according to claim 5, wherein: The volume ratio of the propidium iodide stain solution to the spore suspension is 1:199.

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