Method for quickly detecting latent infection and medication of strawberry anthracnose in seedling stage

A strawberry anthracnose and detection method technology, applied in the prevention and control and early warning of drug resistance, plant disease detection, identification, biological fields, can solve the problems of long detection period, high detection cost, cumbersome electrophoresis process of thermal cycle instruments, etc.

Active Publication Date: 2018-05-29
ZHEJIANG FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The result of this method is accurate and reliable, but it has the characteristics of a long detection period and cannot achieve the purpose of timely monitoring
Based on a series of molecular detection methods such as polymerase chain reaction (PCR), it is also the most common method to amplify specific genes in vitro to detect drug-resistant mutants. Detection requires precise thermal cyclers and tedious electrophoresis processes. The cost is high, and it cannot be tested out of the laboratory environment

Method used

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  • Method for quickly detecting latent infection and medication of strawberry anthracnose in seedling stage
  • Method for quickly detecting latent infection and medication of strawberry anthracnose in seedling stage
  • Method for quickly detecting latent infection and medication of strawberry anthracnose in seedling stage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1, LAMP detection of strawberry anthracnose at seedling stage to the test of strawberry plant samples in the field

[0077] For the collected samples of 20 strawberry plants suspected of suffering from anthracnose, 100mg samples were respectively taken from the shortened stems, stalks, petioles and leaves with diseased symptoms or no obvious lesions, and the above DNA extraction method was used. Strawberry plant tissue DNA was directly extracted by lateral-flow device (LFD). The plant material was lysed in the Extraction buffer, and then the buffer containing the crude extract was added to the release pad of the LFD, and it was chromatographed along the capillary of the device onto the nitrocellulose membrane (LFD membrane). DNA on LFD membranes can be amplified by adding a portion of the membrane directly to the LAMP amplification reaction. Strawberry plant tissue samples (100 mg) were added to a plastic bottle containing five steel balls (5 mm in diameter) an...

Embodiment 2

[0091] Embodiment 2, the accuracy of G143A strawberry anthracnose pathogen LAMP detection

[0092] The DNA of 10 Strawberry Anthracnose strains collected and isolated from different regions was used as a template (extracted with a fungal genome DNA extraction kit) to analyze the accuracy of LAMP detection of anthracnose resistance. Using the different dose method, measure the resistance of the isolated Strawberry anthracnose bacterial strains to the two agents. After each bacterial strain is activated and cultured for 3 days, take the same growing bacteria cake (5mm) on the same growth circle of the petri dish and transfer it to On the PDA medium containing 0, 5, 20 and 100 μg / mL thiophanate-methyl, after culturing for 3 days at 25°C in the dark, the mycelial growth was observed. The different phenotypes of the tested strains can be defined as (S): minimum inhibitory concentration MIC100 μg / mL.

[0093] The detection results of 10 strains were: 5 strains were highly resistant...

Embodiment 3

[0106] Embodiment 3, the sensitivity of LAMP detection

[0107] For the LAMP detection sensitivity test of strawberry anthracnose at the seedling stage, the DNA template of G. -7 ng / μL, respectively using it as a template for LAMP and PCR amplification.

[0108] PCR amplification uses primers TU-F3 and TU-B3, the reaction system is 25 μL: 12.5 μL 2×PCR Master, 0.4 μM primers Hcytb-F and Hcytb-F, 1 μL DNA template, dd H2O to make up 25 μL, the PCR program is 95 °C 5min; 35 cycles of 95°C for 30s, 55°C for 30s and 72°C for 30s, followed by 72°C extension for 10min.

[0109] PCR can be carried out with reference to the method described in Detection and characterization of benzimidazole resistance of Botrytis cinerea in greenhouse vegetables published in European Journal of Plant Pathology.

[0110] The amplification system and procedure of LAMP are the same as in Example 1.

[0111] Comparing the sensitivity of LAMP and PCR, the results show that the concentration of DNA templ...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) detection method for detecting strawberry anthracnose in the seedling stage and an LAMP primer used in the method; and the method can be used for quickly detecting whether strawberry seedlings carry germs or not on the site. The invention also discloses an LAMP detection method for detecting anti-QoI germicide G143A genotype strawberry colletotrichum gloeosporioides and an LAMP primer used in the method; and the method can be used for specifically detecting strawberry colletotrichum gloeosporioides with high resistance toQoI germicides. The method disclosed by the invention has the characteristics of simplicity, quickness, low cost, high sensitivity and the like, can greatly improve the detection efficiency, and has important practical significance for seedling stage detection and drug resistance epidemic early warning of strawberry anthracnose.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a loop-mediated isothermal amplification (LAMP) primer set for detecting bacteria-carrying bacteria in seedling-stage strawberry plants and anti-QoI fungicides of G. fragariae anthracnose and a method for using the same, and belongs to the detection, identification, and prevention of plant diseases and the technical field of early warning of drug resistance. Background technique [0002] Strawberry (Fragaria ananassa), Rosaceae, Fragaria, is a perennial perennial herbaceous plant, which belongs to berry fruit in horticulture. Among the various berries in the world, the cultivated area and yield of strawberry are second only to grapes, ranking second. Strawberry is favored by producers and consumers because of its high yield, quick effect, and rich protein and vitamins. In recent years, China's strawberry planting area has grown rapidly. In 2015, China became the largest strawberry pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/173C12Q2565/125
Inventor 张传清胡小然戴德江吴鉴艳
Owner ZHEJIANG FORESTRY UNIVERSITY
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