Molecular diagnosis method for gray mold

A technique for botrytis cinerea and botrytis cinerea, which is applied in the field of detection and identification of botrytis cinerea pathogens, can solve problems such as long time period and difficult control, and achieves good early diagnosis, rapid method and good specificity.

Inactive Publication Date: 2006-03-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It is difficult to judge with the naked eye at the initial stage of the onset of gray mold in fields and protected areas. When it is visible to the naked eye, the disease will develop rapidly and be difficult to control. , humidity and experience judgment, give guidance to vegetable f

Method used

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  • Molecular diagnosis method for gray mold
  • Molecular diagnosis method for gray mold
  • Molecular diagnosis method for gray mold

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0048] Example 1. Specificity test

[0049] Weigh 0.03g each of 9 kinds of plant tissues hm1, hm2, hm3, hm4, bf1, bf2, sm1, sm2, LJYM, and add 200μL extraction buffer respectively, after thorough grinding, add 300μL extraction buffer and mix well , 65 ℃ water bath for 10 minutes; add an equal volume of chloroform: isoamyl alcohol (24:1), mix well, place on ice for 10 minutes; centrifuge at 12000 rpm / min for 10 minutes; take the supernatant, add 0.6 times the volume of isopropanol, and mix Homogenize, place at -20°C for 30 min, centrifuge at 12000 rpm / min for 10 min, discard the supernatant; wash the precipitate twice with pre-cooled 70% ethanol, and dry; add 50 μL ddH 2 O dissolves.

[0050] Add 2μL of template, 5μL of 10×PCR buffer, 2.5μL of dNTP (2.0mmol / L), 0.04μL of Taq enzyme (5U / μL), and 0.25μL of forward and reverse primers (20μmol / L) into 9 reaction tubes. , Add ddH 2 O to 50μL total volume.

[0051] PCR amplification was carried out according to the following procedure: 9...

Example Embodiment

[0053] Example 2 Sensitivity test

[0054] According to the ratio shown in Table 1, mix healthy tomato leaves with in vitro cultured Botrytis cinerea hyphae, weigh about 0.03g of plant tissue mixed with a certain amount of Botrytis cinerea hyphae, and extract template DNA according to the same method as in Example 1. Perform PCR amplification, the results are as figure 2 Shown. The results showed that No. 2, 3, and 4 could amplify clear and bright bands, and No. 1 and No. 5 were not amplified.

[0055] Label

[0056] After diluting the No. 4 template in different ratios of 2 times, 5 times, 10 times, 20 times, and 50 times, PCR amplification was performed according to the same method as in Example 1. The result is image 3 Shown. It can be seen that positive amplification can be obtained after diluting 2 times, 5 times, 10 times, 20 times and 50 times, but the amplification efficiency also decreases. When tested under UV transmission, the 20-fold dilution is the lowest a...

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Abstract

A molecular diagnosing method for botrytis of tomato includes such steps as designing a pair of PCR primers specific to the pathogen of tomato botrytis, using them for PCR amplifying of the specimen to be tested, and judging if the tomato plant is suffered from botrytis. Its advantages are high speed, high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of plant disease and insect pest control, in particular to the detection and identification of the gray mold pathogen. Background technique [0002] Botrytis cinerea is a very harmful disease in the vegetable area in northern my country. Grapes, tomatoes, peppers, cucumbers, kidney beans and other vegetables cultivated in protective facilities such as plastic greenhouses, greenhouses, and small sheds often occur and become popular, and in severe cases, the yield can be reduced by 100%. 20% to 30% or more. In the southern vegetable area of ​​my country, this disease rarely occurred in the past, but in recent years, due to the development of protected cultivation, Botrytis cinerea has also begun to occur and has a tendency to increase year by year. [0003] Botrytis mainly damages fruits, but can also damage leaves and stems. Fruit damage generally starts from the remaining petals, receptacles, etc., and moist, gray-br...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447
Inventor 马雅军王娜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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