GB/T 36824-2018 in English
VALIDDetection and identification of Mycosphaerella pini E.Rostrup
- Issued on:2018-09-17
- Implemented on:2019-04-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
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《GB/T 36824-2018松针红斑病菌检疫鉴定方法》由TC271（全国植物检疫标准化技术委员会）归口，主管部门为国家标准化管理委员会。
Introduction
Professional interpretation of quarantine and identification method for pine needle red spot pathogen GB/T 36824—2018
Overview of standard framework
| Chapter number | Chapter content |
|---|---|
| 1 | Scope |
| 2 | Normative references |
| 3 | Basic information on pine needle red spot pathogen |
| 4 | Principle of method |
| 5 | Equipment and reagents |
Detailed explanation of laboratory inspection process
Laboratory inspection is the key link in the quarantine and identification of pine needle red spot pathogen. The following is a detailed operation process:
- Sampling method: Sampling is carried out in accordance with SN/T 2122 standard.
- On-site inspection: Check whether the pine needles have light green, yellow to reddish brown spots, and whether necrosis and shedding occur.
- Cut section inspection: Use a perspective microscope to observe the characteristics of ascospores, ascospores or conidiophores.
- Isolation and culture: Colony culture was carried out on malt extract agar (MEA) plates, with the optimum temperature of 20℃ and pH value of 3-5.
Comparison of pathogen morphological characteristics
| Project | Pine needle red spot pathogen | Pine needle brown spot pathogen | Pine needle brown blight pathogen |
|---|---|---|---|
| Conidia | Colorless, thin and smooth wall. Size is (12μm～48μm)×(2μm～3μm) | Dark brown or smoky brown, with thick wall and black wart-like protrusions. | Light yellowish brown, thin and smooth wall. |
| Culture characteristics | The colony grows slowly and is gray to black. | The colony grows faster and has olive green spore mucus. | The colony is gray and fluffy and grows fastest. |
Real-time fluorescence PCR detection
Real-time fluorescence PCR detection is an important means to identify pine needle red spot pathogens. Through the fluorescence quantitative PCR instrument to amplify specific DNA sequences, it is determined whether the sample carries the pathogen. The key steps include:
- Primer probe sequences: MP-F, MP-R and MP-P are used for specific amplification.
- Reaction conditions: 95℃ pre-denaturation for 30 seconds, 40 cycles (95℃ for 15 seconds, 60℃ for 25 seconds).
Implementation recommendations
To ensure effective quarantine of pine needle red spot pathogen, the following measures are recommended:
- Strengthen on-site inspections, especially for imported seedlings and needles.
- During laboratory inspections, fully combine microscopic observation and molecular detection technology to improve identification accuracy.
- Regularly update quarantine standards to respond to possible technological evolution of pathogens.
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