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Early stage rapid molecular detection method for rape sclerotinia rot

A technology for sclerotinia sclerotiorum and molecular detection of rapeseed is applied in the field of molecular detection and detection of plant diseases, which can solve the problems of large workload, time-consuming and labor-intensive, difficult to predict and forecast, and achieve the effect of high sensitivity and rapid detection.

Inactive Publication Date: 2009-05-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional microbiological detection and identification method needs to go through pathogen isolation, cultivation and classification of biological characteristics, which is time-consuming and labor-intensive, with a large workload, and it is not much ahead of the development of field diseases, so it is difficult to apply to production forecasting

Method used

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  • Early stage rapid molecular detection method for rape sclerotinia rot
  • Early stage rapid molecular detection method for rape sclerotinia rot
  • Early stage rapid molecular detection method for rape sclerotinia rot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: detect the specificity test of the specific primer designed by the present invention to sclerotinia and other fungal isolates

[0059] 1. Culture of fungal isolates and fungal hyphae

[0060] See Table 1 for a list of S. sclerotiorum and other fungal isolates and their collection locations. Inoculate the fungal isolate on the PDA medium (formula and its preparation method: use the conventional method, i.e. cook with 200g of peeled potatoes, 20g of glucose and a small amount of distilled water, add distilled water to 1000ml), cultivate at 20°C, and activate for 2-3 Second-rate. Then use a puncher with a diameter of 5mm to punch out the agar block containing mycelium, inoculate it on a PDA plate covered with cellophane, scrape an appropriate amount of mycelium after 48 hours, and store it at -20°C.

[0061] Table 1 The collection locations and molecular detection results of the isolates to be tested

[0062]

[0063]

[0064] The above-mentioned iso...

Embodiment 2

[0076] Embodiment 2: detect the sensitivity test of the specific primer designed by the present invention to sclerotinia

[0077] 1. Culture of Sclerotinia strain SUN-F-M mycelia

[0078] The Sclerotinia strain SUN-F-M mycelium was inoculated on the PDA medium (refer to the above "Summary of the Invention" for the formula), cultured at 20° C., and activated 2-3 times. Then use a puncher with a diameter of 5 mm to punch out agar blocks containing mycelium, inoculate them on a PDA (formulation as described above) plate covered with cellophane, scrape an appropriate amount of mycelium after 24 hours and freeze at -20°C.

[0079] 2. Extract the total DNA of Sclerotinia strain SUN-F-M

[0080] With reference to Sambrook et al. (Sambrook, J., Frisch, E.F., Maniatis, T., Molecular Cloning: A Laboratory Manual, second ed.1989.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), the specific steps are as follows: weigh 0.2g The mycelium frozen at -20°C was fully ground into powder...

Embodiment 3

[0100] Example 3: Specific steps for implementing rapid molecular detection of early onset of Sclerotinia sclerotiorum in field

[0101] 1. Gather rapeseed petals

[0102] Sterilize the 1.5ml Eppendorf tube first. The rapeseed field of Huazhong Agricultural University in Wuhan City, Hubei Province, China to be tested was divided into 4×4=16 plots, and each plot adopted a 5-point sampling method, and the total number of samples was 80. Littered petals were collected from canola leaves, and one petal was packed into an Eppendorf tube. Store in -80°C freezer.

[0103] 2. Extraction of total DNA from rapeseed petals

[0104] (1) Take out the Eppendorf tube containing the petals from the -80°C refrigerator, and quickly add 200 μl of extraction buffer (recipe is shown in the aforementioned "Summary of the Invention") to submerge the petals;

[0105] (2) Heating with a low-grade heat of a domestic microwave oven, the processing time and sequence are 20s, 20s, 15s, preferably with...

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Abstract

The invention belongs to the plant diseases molecular detection field and relates to the early rapid molecular detection method of sclerotinia rot of colza. The invention is characterized by comprising the collection of weazen rapeseed petals, extraction of total DNA of rapeseed petals, nest-PCR, PCR amplification product analysis, agarose gel electrophoresis and result judgement. If sclerotinia sclerotiorum exists in a sample, a 292bp purpose bank may appear in the agarose gel under ultraviolet light, or else, the sclerotinia sclerotiorum does not exist. The primers of the invention can not only distinguish common plant leaf pathogenic fungi of distance relationship and different categories, such as alternaria genus alternaria sp, fusarium genus fusarium graminearum and coniothyrium genus sclerotinia sclerotiorum sclerotia coniothyrium parasitic fungi, but also can distinguish various fungi of common plant leaf fungal pathogen botrytis also belonging to ascomycotina, discomycete and helotiales with the sclerotinia species; the invention has high sensitivity, can detect the sclerotinia sclerotiorum DNA with the limit of 1fg / Mul, is rapid, and can obtain accurate detection result within 7 hours, however, at least two days or more than 1 week is needed for obtaining the detection result by using the traditional microbiology method.

Description

technical field [0001] The invention belongs to the field of molecular detection and detection of plant diseases, and in particular relates to an early rapid molecular detection method for rape sclerotinia. Background technique [0002] Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum (Lib.) de Bary is the primary disease in rape production in my country, especially in the vast rape planting areas of the Yangtze River Basin. In general, the incidence rate of rapeseed sclerotinia is 10%-30%, and in severe cases, the incidence rate will reach 80%, resulting in a 10%-70% reduction in rapeseed output and a 1%-5% reduction in oil content, causing hundreds of millions of farmers. dollars of economic loss. In recent years, with the change of rapeseed cultivation system, such as the application of no-tillage method, the overwintering survival rate of sclerotia has increased; the large-scale promotion of "double-low" rapeseed has resulted in a single cultivar and reduced ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 付艳苹覃黎谢甲涛姜道宏
Owner HUAZHONG AGRI UNIV
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