Some standard content:
ICS65.020.01
CCS B16
National Standard of the People's Republic of China
GB/T40252—2021
Viability test of Monilinia fruticola (G.Winter) Honey2021-05-21Release
State Administration for Market Regulation
National Standardization Administration
2021-12-01Implementation
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GB/T40252—2021
This document is drafted in accordance with the provisions of GB/T1.12020 Guidelines for Standardization Work Part 1: Structure and Drafting Rules for Standardization Documents.
Please note that some contents of this document may involve patents: The issuing agency of this document does not assume the responsibility for identifying patents. This document is proposed and managed by the National Technical Committee for Plant Quarantine Standardization (SAC/TC271). The drafting units of this document are: Shenzhen Customs of the People's Republic of China, Shenzhen Institute of Inspection and Quarantine, Shenzhen Xianhu Botanical Garden, and the main drafters of this document are: Zhang Jiaming, Gong Ying, Gao Ruifang, Cheng Yinghui, Zhu Liaoyin, Li Na, Huang Heqing, and Liu Rong. rrKaeerkAca-
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1Scope
Activity detection method for brown rot pathogen of American and Australian drupes GB/T40252—2021
This document describes the method for activity detection of the brown rot pathogen of Guan-Australian drupes［Mnniliniafruticolu（G.Witer）Honey］ using ordinary fluorescence microscope or laser scanning confocal microscope: This document is applicable to Guan-Australian drupes pathogen of Fungal species of Guan Detection of conidial viability of American and Australian stone fruit brown rot pathogens 2 Normative references
The contents of the following documents constitute the essential terms of this document through normative references in the text. Among them, for referenced documents with dates, only the version corresponding to that date is applicable. For referenced documents without dates, the latest version (including all amendments) is applicable to this document.
SN/T2589 Detection specification for plant pathogenic fungi 3 Terms and definitions
The following terms and definitions are applicable to this document. 3.1
viability
The property of having vitality.
Econidialviability
Conidial viability
Conidial viability is the property of having vitality. Note: The spores are active, which means the spores are alive; the spores are inactive, which means the spores are dead. 4 Basic Information
Chinese name: Guan Australian type stone fruit brown rot fungus
English name: pathogen of American brown rot of stonefruit Scientific name: Moniliniafructicola (G.Winter) Honcy Former name: Sclerotinia fruclicolu (Winter) Rehm Asexual MoniliafructicolaBatra Classification status: Belongs to Fungi, Ascomycota, Ascomycota, Helotiales. Sclerotiniaceae. Monitinia genus. The host, symptoms, colony characteristics and conidia morphological characteristics of the American and Australian type of stone fruit brown rot fungus are shown in Appendix A. 5 Principle
Use fluorescein monoacetate (Flourcsceiniacctatc.FA) and propidium iodide (Propidium iodide, PI fluorescent dye 1
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GB/T40252—2021
to dye the spores of the Australian type of stone fruit brown rot fungus. According to FDA, it can remain in the living cells through cell metabolism, making the active spores emit green fluorescence. If the cell membrane is damaged, the fluorescein is lost, so that the inactive spores do not emit fluorescence; PI can penetrate the dying or already Dead cell membranes make inactive spores emit red fluorescence, while PI cannot penetrate active cell membranes, making active spores non-fluorescent. Based on the above characteristics, ordinary light-extinguishing microscope or laser scanning confocal microscope is used to detect the activity of pathogen conidia. Analyze the life and death of conidia. 6 Instruments and reagents
Instruments
Ordinary fluorescence microscope or laser scanning confocal microscope, ultra-clean workbench, flat bed, high-pressure sterilization pot, biochemical incubator, refrigerator, centrifuge.
6.2 Reagents
Flourescein Diacetate (FDA): Weigh 0.1g FDA powder, add 10ml acetone, and prepare a concentration of 10m g·mL- mother solution. Store in a brown bottle at 4℃ and away from light. Dilute with acetone before use to prepare a 0.5mgmL- working solution.
Propidlium Iodide (PI): Weigh 0.01g PI powder, add 10mL sterile water, and prepare a mother solution with a concentration of 1mg·mL. Store in a brown bottle at 1℃ and away from light. Dilute with sterile water before use to prepare a 0.05mg·ml. working solution. 6.3 Main culture medium
Potato inulin agar medium (PDA): 200g peeled potatoes are cut into small cubes, added with 1000mL of white water, and boiled for 15min. Filter with four layers of gauze to obtain the filtrate, and add 18g inulin and 18g agar into the filtrate. Powder, dissolve in water and dilute to 1000 ml. Dispense into Erlenmeyer flasks. Sterilize with high pressure steam for 20 min. 7. Activity detection method
7.1 Preparation of test samples
Scrape the spores of the host with symptoms and put them into a centrifuge with sterile water to prepare a spore suspension with a final concentration of 10 to 100 spores per microliter. Microscopic examination, set aside. 7.2 Spore staining
7.2.1 FDA staining
Take 199L of spore suspension, add 1μL of FDA with a mass concentration of 0.5mg·mL-, mix, and light stain at room temperature for 15min. Centrifuge at 16000g for 1min, fill with supernatant to stop staining, add sterile deionized water to wash once, and resuspend. Prepare and use immediately, and place away from light.
7.2.2PI staining
Take 199μl of spore suspension and add 1μL of PI with a mass concentration of 0.05 mg·mL, stain at room temperature in the dark for 4min, centrifuge at 16 000g for [min], discard the supernatant to stop staining, wash once with sterile deionized water, resuspend, prepare and use immediately. Keep away from light.
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7.3 Activity detection by ordinary fluorescence microscope or laser scanning confocal microscope 7.3.1 Ordinary fluorescence microscope detection
7.3.1.1 Slide preparation
Pipette 5μL of the sample treated with activity dye to prepare glass slides and seal the slides. 7.3.1.2 Detection
GB/T 40252—2021wwW.bzxz.Net
Place the prepared tegument immediately on the stage of an ordinary fluorescence microscope, find the spores under a low-power microscope, then switch to a 40x microscope for observation, and fine-tune until the image in the field of view is clear and observe and count. Detect at least 30 spores and observe the staining. FA staining detection: turn on the FA light path, set the excitation wavelength of the FA channel to 488nm, collect the emission wavelength of the extinction signal to 530nm, and collect the fluorescence signal: fine-tune until the image in the field of view is clear: (see Appendix B) PI staining detection: turn on the fluorescence light path. Set the excitation wavelength of the flammability channel to 534nm, collect the emission wavelength of the fluorescence signal to 617nm, and collect the fluorescence signal. Fine-tune until the image in the field of view is clear. (See Appendix B) Note: In order to ensure that the image can reflect the most realistic fluorescent staining of the spores, it is particularly necessary to observe whether the spore image obtained in the bright field channel is clear. 7.3.2 Laser scanning confocal microscope activity detection 7.3.2.1 Preparation
Absorb 5. Prepare slides for samples treated with active dyes and seal the slides. 7.3.2.2 Laser setting
Select the corresponding laser tube (argon ion laser) to excite the fluorescence signal. For FID)A detection, set the excitation wavelength of the fluorescence channel (488nm), collect the emission wavelength of the fluorescence signal (530nm), and set a bright field channel as a control. For PI detection, set the excitation wavelength of the fluorescence channel (534nm), collect the emission wavelength of the fluorescence signal (617nm), and set a bright field channel as a control. 7.3.2.3 Scanning settings
Select low-pixel scanning mode (xy: 512×512), repeat scanning times Avtragc is 1 time, scanning speed Spccd is 9, adjust the detection pinhole Pinhole, photomultiplier tube gain Gain and laser scanning intensity Scan Str according to the imaging effect, and adjust the image to a better quality effect. Then use the precise deduction mode (xy: 2018×2018) and then adjust the scanning mode according to the signal-to-noise ratio. Select the precise pixel flat scanning mode for rough scanning (xy: 2018×2048), repeat scanning times Avcragc is 2 times. Scanning speed Spccdl is 6 to obtain the final image.
7.3.2.4 Detection
Immediately place the prepared fragments on the laser scanning confocal microscope stage 1, find the spores under the low-power microscope, switch to the 40-power microscope for observation, fine-tune until the image is clear in the field of view, scan at least 30 spores, and observe the staining results. (See Appendix () Note: To ensure that the image can reflect the most realistic carbon light staining of the spores, it is particularly necessary to observe whether the spore image obtained under the auxiliary field channel is clear. 8 Result judgment and expression
Use ordinary carbon light microscope or laser confocal microscope to detect at least 30 conidia treated with FDA and PI respectively. The result judgment and expression are as follows:
If the conidia detected by FIA treatment emit green fluorescence, and the conidia detected by PI treatment do not emit fluorescence, then the conidia are judged to be green fluorescence. The conidia are active;
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GB/T40252—2021
If the conidia detected by PI treatment emit red fluorescence, and the conidia detected by FDA treatment do not emit fluorescence, then the conidia are judged to be inactive.
Sample and original data set preservation
9.1 Sample preservation
Whether the sample is stored depends on the state of the sample. The 6 slices should be well preserved: If active The samples of Australian stone fruit rot should be stored in a slanted manner. For example, if the trade dispute is a Shar-Pe trade dispute, the samples should be stored until the dispute is resolved. After the storage period expires, the samples should be sterilized. The sample storage method should comply with the provisions of SV/I2589. 9.2
Original data storage
After the sample test is completed, the original record sheet and test results or certificates should be filed and kept in a safe place for re-testing, negotiation and arbitrationrrKaeerkAca-
A.1 Main hosts
Appendix A||t t||(Informative)
GB/T40252—2021
Hosts, symptoms, colony characteristics and conidia morphology of American and Australian type stone fruit brown rot pathogens The main hosts are apple (Malus domestica), papaya (Caricapupaya), European plum (Cerasus Humilis), cherry (Cerus pseudocerasus), European dogwood (Cornusmas), apricot (Prunus armeniacu), European citrus cherry (Prunus auium), European sour cherry (Prunus Cerasus), European plum (Prunus domestica), almond (Prunus dulcis, peach (Prunus persica), plum (Prunus saticina), apricot plum (Prunus simonii), grape arrow (Vitis uinifera), loquat (Eriobotrya japonica). The pathogen can also damage hosts such as Chaenomeles, Crataegus, Cvdonia, Eriohorrya, Phyllostachys, Prunus, and Pyrus. A.2 Symptoms of damage
The disease mainly damages fruits. In the early stage of the disease, gray-brown circular lesions form on the fruit skin, and then the lesions quickly spread to the whole fruit, and the flesh turns brown and soft rots. A scattered gray-brown fluffy layer is produced on the surface of the diseased part. Finally, most or all of the diseased fruits rot and fall off, or shrink into occasional fruits that hang on branches for a long time without falling off. If under low humidity conditions, the whole fruit shrinks and becomes dry. Infected flowers and leaves turn brown and wilt. A typical wilting pattern is formed, with brown sunken areas (ulcer spots) on the stems, usually with gum accumulated on the surface. Under humid conditions, conidiophores are produced on these infected tissues. Symptoms of the Australian-type drupe brown rot are shown in Figure A.1. a) Symptoms of the American-Australian-type drupe brown rot on apples b) Symptoms of the American-Australian-type drupe brown rot on plums c) Symptoms of the American-Australian-type drupe brown rot on peaches Figure A.1 Symptoms of the American-Australian-type drupe brown rot Figure A.3: Colony characteristics
The colony grows quickly. It is cultured on PDA medium at 22°C for about 8 days. The colonies can cover the entire culture medium IIIL in concentric ring patterns. The colony edges are complete and the texture is uniform. The colonies are well developed and velvety. The colony color is gray at the initial stage. The phylum is gray-yellow or gray-brown in the later stage, with abundant production and conidia piles on the colony surface in concentric rings. The colony morphology of the strain grown on PDA medium is shown in Figure A.25
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GB/T40252—2021
A.4 Conidia morphological characteristics
The colony morphology of the strain grown on PDA
The spore chain is rosary-shaped, lemon-shaped to oval, and sometimes half of the top. The size is (8um28um) × (5μm~19pm), colorless and transparent, and gray-yellow when aggregated. The conidiophores are short and branched or unbranched, with conidia borne at the top. 6
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Appendix B
(Informative)
Observation results of spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. The staining effect of FDA on spores of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.110μm
Explanation of index numbers:
A, B——Staining results of living spores, A is the fluorescence channel, B is the bright field: (D)——Staining results of dead spores, for the fluorescence channel, D) is the bright field GB/T40252—2021
Observation results of FDA on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. Figure B.1
Observation results of PI on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.2. -riKaeerka
GB/T40252—2021
Index number description:
Dead spore staining results, A is the fluorescence channel, B is the bright field: A. B
Live spore staining results, C is the fluorescence channel, D is the bright field. Figure B.2
Ordinary fluorescence microscope observation of the staining effect of the spores of the American and Australian type of stone fruit brown rot fungus-rKaeerkca-1. Sample preservation
Whether the sample is stored or not, the sample should be stored in a corresponding way according to its state. It should be stored for 6 days: if active Australian stone fruit rot is found, it should be stored obliquely. If the dispute is not resolved, the sample should be stored until the dispute is resolved. After the storage period expires, the sample should be sterilized. The sample storage method should comply with the provisions of SV/I2589. 9.2
Original data preservation
After the sample test is completed, the original record sheet and test results or certificates shall be filed and properly kept for re-testing, negotiation and arbitrationrrKaeerkAca-
A.1 Main hosts
Appendix A
(Informative)
GB/T40252—2021
Hosts, symptoms, colony characteristics and conidia morphological characteristics of American and Australian type stone fruit brown rot pathogens The main hosts are apple (Malus domestica), papaya (Caricapupaya), European plum (Cerasus Humilis), cherry (Cerus pseudocerasus), European dogwood (Cornusmas), apricot (Prunus armeniacu), European mandarin cherry (Prunus auium), European sour cherry (Prunus Cerasus), European plum (Prunus domestica), almond (Prunus dulcis), peach (Prunus persica), plum (Prunus saticina), apricot plum (Prunus simonii), grape arrow (Vitis uinifera), loquat (Eriobotrya japonica). The pathogen can also cause damage to hosts such as Chaenomeles, Crataegus, Cvdonia, Eriohorrya, Phllostachvs, Prunus, and Pyrus. A.2 Symptoms
The disease mainly harms fruits. In the early stage of the disease, gray-brown circular spots form on the fruit coat. Then the spots quickly spread to the whole fruit, and the flesh turns brown and soft. A scattered gray-brown fluffy layer appears on the surface of the diseased part. Finally, most or all of the diseased fruits rot and fall off, or shrink into occasional fruits hanging on branches for a long time. If under low humidity conditions, the whole fruit shrinks and becomes dry. The infected flowers and leaves turn brown and wilt. A typical wilting shape is formed, and brown sunken areas (ulcer spots) are formed on the stems. Usually, gum accumulates on the coat. Under humid conditions, gum is produced on these infected tissues. The symptoms of the Australian-type drupe brown rot pathogen are shown in Figure A.1. a) Symptoms of the American-Australian-type drupe brown rot on apples b) Symptoms of the American-Australian-type drupe brown rot on plums c) Symptoms of the American-Australian-type drupe brown rot on peaches Figure A.1 Symptoms of the American-Australian-type drupe brown rot pathogen Figure A.3: Colony characteristics
The colony grows fast. It is cultured on PDA medium at 22℃ for about 8 days. The colony can cover the entire culture medium IIIL in a concentric ring pattern. The colony edge is complete, the texture is uniform, the gastrocnemius is well developed, and it is in the shape of a floc. The colony color is gray in the early stage, and grayish yellow or gray in the later stage. The yield is abundant and the colony surface is The conidia are piled in concentric rings. The colony morphology of the strain growing on PDA medium is shown in Figure A.25
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GB/T40252—2021
A.4 Conidia morphological characteristics
The colony morphology of the strain growing on PDA
The spore chain is bead-like, lemon-shaped to oval, and sometimes half-cut at the top. The size is (8um28um)×(5μm~19pm), colorless and transparent, and grayish yellow when aggregated. The conidiophores are short and branched or unbranched, with conidia at the top, 6|| tt||-riKaeerkca-
Appendix B
(Informative)
Observation results of spore staining of American and Australian stone fruit brown rot fungus under ordinary fluorescence microscope. The staining effect of FDA on spore staining of American and Australian stone fruit brown rot fungus under ordinary fluorescence microscope is shown in Figure B.110μm
Indication number description:
A, B——Staining results of living spores, A is the fluorescence channel, B is the bright field: (D)——Staining results of dead spores, for the light channel, D) is the bright field GB/T40252—2021
Observation of FDA under ordinary fluorescence microscope Figure B.1: Staining effect of PI on spores of brown rot fungus of American and Australian drupes
Observation of PI on spores of brown rot fungus of American and Australian drupes under ordinary fluorescence microscope. See Figure B.2. -riKaeerka
GB/T40252—2021
Indication number description:
Results of dead spore staining, A is the fluorescence channel, B is the bright field: A. B
Results of live spore staining, C is the fluorescence channel, D is the bright field. Figure B.2: Staining effect of PI on spores of brown rot fungus of American and Australian drupes under ordinary fluorescence microscope -rKaeerkca-1. Sample preservation
Whether the sample is stored or not, the sample should be stored in a corresponding way according to its state. It should be stored for 6 days: if active Australian stone fruit rot is found, it should be stored obliquely. If the dispute is not resolved, the sample should be stored until the dispute is resolved. After the storage period expires, the sample should be sterilized. The sample storage method should comply with the provisions of SV/I2589. 9.2
Original data preservation
After the sample test is completed, the original record sheet and test results or certificates shall be filed and properly kept for re-testing, negotiation and arbitrationrrKaeerkAca-
A.1 Main hosts
Appendix A
(Informative)
GB/T40252—2021
Hosts, symptoms, colony characteristics and conidia morphological characteristics of American and Australian type stone fruit brown rot pathogens The main hosts are apple (Malus domestica), papaya (Caricapupaya), European plum (Cerasus Humilis), cherry (Cerus pseudocerasus), European dogwood (Cornusmas), apricot (Prunus armeniacu), European mandarin cherry (Prunus auium), European sour cherry (Prunus Cerasus), European plum (Prunus domestica), almond (Prunus dulcis), peach (Prunus persica), plum (Prunus saticina), apricot plum (Prunus simonii), grape arrow (Vitis uinifera), loquat (Eriobotrya japonica). The pathogen can also cause damage to hosts such as Chaenomeles, Crataegus, Cvdonia, Eriohorrya, Phllostachvs, Prunus, and Pyrus. A.2 Symptoms
The disease mainly harms fruits. In the early stage of the disease, gray-brown circular spots form on the fruit coat. Then the spots quickly spread to the whole fruit, and the flesh turns brown and soft. A scattered gray-brown fluffy layer appears on the surface of the diseased part. Finally, most or all of the diseased fruits rot and fall off, or shrink into occasional fruits hanging on branches for a long time. If under low humidity conditions, the whole fruit shrinks and becomes dry. The infected flowers and leaves turn brown and wilt. A typical wilting shape is formed, and brown sunken areas (ulcer spots) are formed on the stems. Usually, gum accumulates on the coat. Under humid conditions, gum is produced on these infected tissues. The symptoms of the Australian-type drupe brown rot pathogen are shown in Figure A.1. a) Symptoms of the American-Australian-type drupe brown rot on apples b) Symptoms of the American-Australian-type drupe brown rot on plums c) Symptoms of the American-Australian-type drupe brown rot on peaches Figure A.1 Symptoms of the American-Australian-type drupe brown rot pathogen Figure A.3: Colony characteristics
The colony grows fast. It is cultured on PDA medium at 22℃ for about 8 days. The colony can cover the entire culture medium IIIL in a concentric ring pattern. The colony edge is complete, the texture is uniform, the gastrocnemius is well developed, and it is in the shape of a floc. The colony color is gray in the early stage, and grayish yellow or gray in the later stage. The yield is abundant and the colony surface is The conidia are piled in concentric rings. The colony morphology of the strain growing on PDA medium is shown in Figure A.25
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GB/T40252—2021
A.4 Conidia morphological characteristics
The colony morphology of the strain growing on PDA
The spore chain is bead-like, lemon-shaped to oval, and sometimes half-cut at the top. The size is (8um28um)×(5μm~19pm), colorless and transparent, and grayish yellow when aggregated. The conidiophores are short and branched or unbranched, with conidia at the top, 6|| tt||-riKaeerkca-
Appendix B
(Informative)
Observation results of spore staining of American and Australian stone fruit brown rot fungus under ordinary fluorescence microscope. The staining effect of FDA on spore staining of American and Australian stone fruit brown rot fungus under ordinary fluorescence microscope is shown in Figure B.110μm
Indication number description:
A, B——Staining results of living spores, A is the fluorescence channel, B is the bright field: (D)——Staining results of dead spores, for the light channel, D) is the bright field GB/T40252—2021
Observation of FDA under ordinary fluorescence microscope Figure B.1: Staining effect of PI on spores of brown rot fungus of American and Australian drupes.
Observation of PI on spores of brown rot fungus of American and Australian drupes by ordinary fluorescence microscope. See Figure B.2. -riKaeerka
GB/T40252—2021
Indication number description:
Results of dead spore staining, A is the fluorescence channel, B is the bright field: A. B
Results of live spore staining, C is the fluorescence channel, D is the bright field. Figure B.2: Staining effect of PI on spores of brown rot fungus of American and Australian drupes by ordinary fluorescence microscope. -rKaeerkca-It is grayish yellow when aggregated. The conidiophores are short and branched or unbranched, with conidia borne at the top. 6
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Appendix B
(Informative)
Observation results of spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. The staining effect of FDA on spores of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.110μm
Explanation of index numbers:
A, B——Staining results of living spores, A is the fluorescence channel, B is the bright field: (D)——Staining results of dead spores, for the fluorescence channel, D) is the bright field GB/T40252—2021
Observation results of FDA on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. Figure B.1
Observation results of PI on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.2. -riKaeerka
GB/T40252—2021
Index number description:
Dead spore staining results, A is the fluorescence channel, B is the bright field: A. B
Live spore staining results, C is the fluorescence channel, D is the bright field. Figure B.2
Ordinary fluorescence microscope observation of the staining effect of the spores of the American and Australian type of stone fruit brown rot fungus-rKaeerkca-It is grayish yellow when aggregated. The conidiophores are short and branched or unbranched, with conidia borne at the top. 6
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Appendix B
(Informative)
Observation results of spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. The staining effect of FDA on spores of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.110μm
Explanation of index numbers:
A, B——Staining results of living spores, A is the fluorescence channel, B is the bright field: (D)——Staining results of dead spores, for the fluorescence channel, D) is the bright field GB/T40252—2021
Observation results of FDA on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope. Figure B.1
Observation results of PI on spore staining of American and Australian drupe brown rot pathogen under ordinary fluorescence microscope is shown in Figure B.2. -riKaeerka
GB/T40252—2021
Index number description:
Dead spore staining results, A is the fluorescence channel, B is the bright field: A. B
Live spore staining results, C is the fluorescence channel, D is the bright field. Figure B.2
Ordinary fluorescence microscope observation of the staining effect of the spores of the American and Australian type of stone fruit brown rot fungus-rKaeerkca-
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